plant physiology preview. published on august 12, …...2015/08/12 · type ii system is the most...

Cas9-gRNA directed genome editing in maize

1

1

Cas9-gRNA directed genome editing in maize 2

3

4

5

Corresponding Author A Mark Cigan DuPont Pioneer 7300 NW 62nd Ave Johnston 6

IA 50131 USA 7

Telephone 1-(515)-535-3904 8

E-mail MarkCiganPioneercom 9

10

11

Research Area Breakthrough Technologies 12

13

Plant Physiology Preview Published on August 12 2015 as DOI101104pp1500793

Copyright 2015 by the American Society of Plant Biologists

httpsplantphysiolorgDownloaded on November 7 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved


2

Targeted Mutagenesis Precise Gene Editing and Site-Specific Gene 14

Insertion in Maize Using Cas9 and Guide RNA 15

16

Sergei Svitashev1 Joshua K Young1 Christine Schwartz Huirong Gao S Carl Falco 17

and A Mark Cigan 18

19

Trait Enabling Technologies 20

DuPont Pioneer 7300 NW 62nd Avenue Johnston Iowa 50131 USA 21

22

23

24

25

Summary 26

27

Advances in double-strand break technologies modernize genome editing in maize28



3

Footnotes 29

1 These authors contributed equally to the article 30

Address correspondence to MarkCiganPioneercom 31

32

The author responsible for distribution of materials integral to the findings presented in 33

this article in accordance with the Journal policy described in the Instructions for 34

Authors (httpwwwplantphysiolorg) is Mark Cigan (MarkCiganPioneercom) 35

36

AMC and SCF conceived the project SS JKY HG and AMC designed the 37

experiments SS JKY and CS performed the experiments SS JKY CS and 38

AMC analyzed the data SS and AMC wrote the article 39

40



4

41

ABSTRACT 42

Targeted mutagenesis editing of endogenous maize genes and site-specific insertion 43

of a trait gene using Cas9-guide RNA technology is reported in Zea mays DNA vectors 44

expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and 45

single guide RNAs were co-introduced with or without DNA repair templates into maize 46

immature embryos by biolistic transformation targeting five different genomic regions 47

upstream of the liguleless-1 gene (LIG) male fertility genes (MS26 and MS45) and 48

acetolactate synthase genes (ALS1 and ALS2) Mutations were subsequently identified 49

at all sites targeted and plants containing biallelic multiplex mutations at LIG MS26 and 50

MS45 were recovered Biolistic delivery of guide RNAs (as RNA molecules) directly into 51

immature embryo cells containing pre-integrated Cas9 also resulted in targeted 52

mutations Editing the ALS2 gene using either single-stranded oligonucleotides or 53

double-stranded DNA vectors as repair templates yielded chlorsulfuron resistant plants 54

Double-strand breaks generated by RNA guided Cas9 endonuclease also stimulated 55

insertion of a trait gene at a site near liguleless-1 by homology-directed repair Progeny 56

demonstrated expected Mendelian segregation of mutations edits and targeted gene 57

insertions The examples reported in this study demonstrate the utility of Cas9-guide 58

RNA technology as a plant genome editing tool to enhance plant breeding and crop 59

research needed to meet growing agriculture demands of the future 60

61



5

INTRODUCTION 62

The evolution of plant breeding one of the first scientific activities known in the 63

history of human kind has allowed rapid progress of our civilization and continues to be 64

important for the improvement of crops and sustainability of agriculture Traditional 65

breeding however is limited by the starting genetic diversity With the advent of 66

modern molecular biology breederrsquos options have been substantially broadened 67

allowing the purposeful transfer of genetic information from either related or diverse 68

organisms into different plant species thus specifically improving agricultural 69

performance The most common methods of DNA delivery particle bombardment and 70

Agrobacterium infection lead to random gene insertions throughout the genome 71

Integration of trait genes into a specific location is considerably more attractive and can 72

be accomplished through homology-directed repair (HDR) While a few publications 73

before 2009 have reported successful direct application of HDR to modify endogenous 74

plant genes this occurs at impractically low frequencies (Halfter et al 1992 Offringa et 75

al 1993 Miao and Lam 1995 Kempin et al 1997 Hanin et al 2001 Terada et al 76

2002 Terada et al 2007) Studies in different organisms including plants have 77

demonstrated that coincident generation of targeted DNA double-strand breaks (DSBs) 78

is central to improving the frequency (up to 1000-fold or more) of gene editing and site-79

specific gene insertion via HDR (Puchta et al 1993 Choulika et al 1995 Smih et al 80

1995 Puchta et al 1996) DSBs in eukaryotic cells can be repaired using two different 81

pathways ndash non-homologous end-joining (NHEJ) and HDR (Roth and Wilson 1986 82

Moore and Haber 1996 Puchta et al 1996 Jasin and Rothstein 2013) The NHEJ 83

pathway is most common in somatic cells and is prone to imperfect repair which results 84

in mutations such as deletions insertions inversions or translocations HDR although 85

less frequent can be directed by the addition of DNA molecules homologous to the 86

DSB region By using repair DNA templates that contain sequence variation or promote 87

the insertion of expression cassettes gene editing and site-specific gene integration can 88

be achieved 89

During the past decade different nucleases capable of generating targeted DSBs 90

were developed and have become important tools to improve gene editing and site-91



6

specific integration in different species including plants (Puchta and Fauser 2014 92

Voytas and Gao 2014 Kumar and Jain 2015) While Zinc-finger nucleases (ZFNs) 93

customized homing endonucleases (meganucleases) and transcription activator-like 94

effector nucleases (TALENs) advanced genome editing the recently discovered Cas-95

guide RNA system has revolutionized the field (Doudna and Charpentier 2014 Hsu et 96

al 2014) This technology emerged from a bacterial adaptive defense system which 97

includes clustered regularly interspaced short palindromic repeats (CRISPR) and 98

CRISPR-associated (Cas) endonuclease which protects the host from plasmid and viral 99

infection (Barrangou et al 2007) Three CRISPR-Cas systems (Type I II and III) with 100

different mechanisms have been described in bacteria and archea (Makarova et al 101

2011 Makarova et al 2011) Type II system is the most simple and requires a single 102

RNA-guided Cas9 protein for recognition and cleavage of invading DNA (Gasiunas et 103

al 2012 Jinek et al 2012) To generate site-specific DSB the system minimally 104

requires Cas9 protein and a duplex of CRISPR RNA (crRNA) a trans-encoded CRISPR 105

RNA (tracrRNA) and the presence of a protospacer adjacent motif (PAM) which flanks 106

the 3rsquo end of the crRNA-targeted sequence (Gasiunas et al 2012 Cong et al 2013) It 107

has been demonstrated that fusion of the crRNA and tracrRNA into a single guide RNA 108

(gRNA) molecule not only simplified the system consisting now of two instead of three-109

components but also significantly improved the frequency of genomic DNA cleavage 110

(Jinek et al 2012 Mali et al 2013) Thus in contrast to ZFNs meganucleases and 111

TALENs which require sophisticated protein engineering to establish specific target site 112

recognition (Smith et al 2000 Arnould et al 2006 Smith et al 2006 Maeder et al 113

2008 Boch et al 2009 Weber et al 2011) Cas9-gRNA reagents are simple to design 114

Targeting of genomic sequences by Cas9-gRNA relies on binding of PAM nucleotides 115

(NGG for S pyogenes) by Cas9 and base-pairing of the gRNA to the adjacent target 116

site sequence 117

The Cas9-gRNA system has been shown to function in many different 118

organisms including plants To date it has been used to generate targeted gene 119

modifications in multiple plant species Arabidopsis thaliana Citrus sinensis Nicotiana 120

benthamiana Nicotiana tabaccum Oryza sativa Solanum lycopersicum Sorghum 121

bicolor Triticum aestivum (for review see Kumar and Jain 2015) Zea mays (Liang et 122



7

al 2014) and Glycine max (Jacobs et al 2015) The majority of these studies 123

however report genome modifications in plants via NHEJ to generate mutations and 124

gene knock-outs While knock-outs are very valuable and allow gene function analysis 125

their application is limited HDR brings other opportunities in genome editing but has 126

been more challenging As a result reports describing successful gene editing or site-127

specific trait gene integration through HDR in plants are limited (Cai et al 2009 Shukla 128

et al 2009 Townsend et al 2009 Ainley et al 2013 DHalluin et al 2013 Li et al 129

2013 Shan et al 2013 Zhang et al 2013) 130

The data presented in this report demonstrate that the Cas9-gRNA system can 131

efficiently facilitate simultaneous multiple gene knockouts native gene editing and site-132

specific gene integration in maize Analysis of T1 plants established that the gene 133

knockouts edits and insertions were heritable and exhibited expected Mendelian 134

segregation This is the first report of Cas9-gRNA technology application in maize 135

demonstrating gene mutagenesis upon direct delivery of gRNA in the form of RNA 136

molecules native gene editing and site-specific gene integration 137

138

139



8

RESULTS 140

141

Targeted Mutations in Maize 142

To test whether maize-optimized RNA-guided Cas9 endonuclease can generate 143

DSBs and alter maize sequences through error-prone NHEJ repair 12 different sites at 144

5 genomic locations were targeted for cleavage (Fig 1 and Supplemental Table S1) 145

These target sequences were selected first by identifying the NGG PAM sequence 146

required for S pyogenes Cas9 and then capturing the 17-24 nucleotides immediately 147

upstream of the PAM sequence for use as the spacer in the gRNA To ensure good U6 148

polymerase III expression and not introduce a mismatch within the gRNA spacer all 149

target sequences contained a G at their 5rsquo end Two of these locations one near 150

liguleless-1 and another within the 5th exon of the maize fertility gene MS26 were 151

previously mutated in corn by custom designed homing endonucleases named LIG34 152

(Gao et al 2010) and Ems26 (Djukanovic et al 2013) respectively To indirectly 153

compare the efficiency of generating DSBs by Cas9-gRNA to these homing 154

endonucleases maize immature embryos were bombarded with DNA vectors 155

containing constitutively expressed meganucleases (LIG34 Ems26) or Cas9 156

components (S pyogenes Cas9 ZmUbiCas9 and gRNAs ZmPolIIIgRNA) and helper 157

genes (ODP2 and Wus) then analyzed by amplicon sequencing for the presence of 158

mutations at the target sites Embryos bombarded with either Cas9 or gRNA DNA 159

expression cassettes served as controls Embryos were harvested 7 days after 160

bombardment total genomic DNA extracted and fragments surrounding the targeted 161

sequences were amplified by PCR Barcoded PCR amplicons were sequenced to an 162

approximate depth of 400000 to 800000 reads aligned to their reference sequence 163

and analyzed It is important to emphasize that during bombardment only a small 164

percentage of embryo cells receive vectors with Cas9 and gRNA expression cassettes 165

while the majority of the cells were not transformed and would be expected to contain 166

unmodified DNA sequences As shown in Table 1 in contrast to controls Cas9-gRNA 167

treated embryos yielded a high frequency of mutations With the exception of one 168

gRNA (MS45-CR1) the majority of the gRNAs tested yielded mutation frequencies 169

greater than 13 Notably embryos transformed with gRNAs that targeted the same 170



9

sequences as LIG34 and Ems26 endonucleases yielded 10- to 20-fold higher mutation 171

frequencies (39 vs 02 and 175 vs 013 respectively) Ten of the most 172

prevalent mutations generated by either the LIG-CR3 gRNA or the LIG34 homing 173

endonuclease are shown in Supplemental Figure S1 In contrast to the predominance 174

of various size deletions present in the sequences derived from the LIG34 175

endonuclease treated embryos and in agreement with previous reports (Feng et al 176

2014) the most common types of mutations promoted by LIG-CR3 guided Cas9 were 177

single nucleotide insertions and single nucleotide deletions Similar results were 178

observed for all other gRNAs used in this study (data not shown) 179

Maize embryos stably transformed by co-bombardment with Cas9 gRNA and 180

selectable and visible marker gene (MoPAT-DsRED) were resistant to treatment with 181

bialaphos due to the presence of the phosphinotricin acetyl transferase gene MoPAT 182

and displayed red fluorescence These traits allowed selection and growth of 183

transformed calli regeneration of fertile plants and evaluation of inheritance of the 184



10

Cas9-gRNA induced mutations describe above in subsequent generations As shown in 185

Table 2 mutations were recovered at all targeted loci when multiple gRNA expression 186

cassettes were introduced either in duplex or triplex with mutant read frequencies 187

similar to those when gRNAs were delivered individually These results demonstrate 188

that the maize codon-optimized RNA-guided Cas9 can simultaneously introduce 189

mutations at multiple loci in a single transformation experiment The frequency of stable 190

event recovery has been reported to be an indicator of cytotoxicity due to off-target 191

recognition for ZFNs in human cell lines and plants (Cornu et al 2007 Szczepek et al 192

2007 Townsend et al 2009) In this multiplex experiment the frequency of 193

establishing callus sectors growing on selection was very similar (more than 1 event per 194

embryo when helper genes are co-bombarded) to the frequency in control experiments 195

(no gRNA delivery) Normal event recovery suggests that for the gRNAs used in this 196

experiment off-target activity was either absent or below a level sufficient to confer 197

detectable cytotoxicity 198

Red fluorescing callus indicative of a stably integrated DsRED gene were used 199

to regenerate plants from these multiplex gRNA experiments Thirty T0 plants (25 200

duplex and 5 triplex) were selected for mutation detection at the target site by copy-201

number determination using qPCR and by direct sequencing of the targeted alleles 202

qPCR analysis revealed that all 30 plants were mutated at the corresponding target 203

sites with a high percentage of plants containing bi-allelic mutations (LIGCas-3 ndash 91 204

MS26Cas-2 ndash 77 MS45Cas-2 ndash 100 Supplemental Table S2) To corroborate the 205

results generated by qPCR DNA fragments spanning these three target sites were 206

amplified by PCR cloned and sequenced from selected plants Sequencing data were 207

in agreement with the qPCR results confirming the high frequency of mutations at all 208

three sites (Table 3) and as expected T0 plants containing bi-allelic mutations at either 209

Ms26 or Ms45 were male sterile (Cigan et al 2001 Djukanovic et al 2013) (data not 210

shown) Plants were also screened by Cas9 and guideRNA-specific PCR 211

(Supplemental Table S3) for the presence of stably integrated copies of Cas9 and 212

gRNA DNA expression cassettes As shown in Table 3 plant 5 did not contain 213

detectable Cas9 and gRNA expression cassettes suggesting that transient delivery of 214

DNA vectors was sufficient to confer biallelic mutations in this experiment Due to this 215



11

observation this plant was selected for transmission studies This plant was fertilized 216

using pollen from wild-type Hi-II plants to produce T1 progeny Examination of these T1 217

plants by qPCR and DNA sequencing demonstrated sexual transmission of parental 218

mutations with expected segregation ratios for all three sites tested (LIGCas-3 ndash 1615 219

[Wt+T] MS26Cas-2 ndash 1412 [Wt+AWt+G] MS45Cas-2 ndash 1413[Wt+TWtdel) 220

Additional plants pollinated with wild-type pollen and containing multiple biallelic gene 221

mutations in the first generation were observed to transmit these mutant alleles to 222

progeny in an expected Mendelian fashion (data not shown) These results 223

demonstrate that RNA-guided Cas9 can simultaneously cleave multiple chromosomal 224

loci and sexually transmit these mutations to progeny 225

226

Transient gRNA Delivery into Cells with Pre-Integrated Cas9 Generates Mutations 227

in Maize 228

As described above co-delivery of DNA vectors containing Cas9 and gRNA 229

expression cassettes into maize embryos yielded heritable mutations However it has 230

been shown in Arabidopsis (Feng et al 2014) and wheat (Wang et al 2014) that 231

progeny plants often do not follow expected Mendelian (11) segregation In the present 232

study segregation distortion was also observed in progeny of two T0 plants (data not 233

shown) One possibility is that stable integration and constitutive expression of gRNAs 234

and Cas9 endonuclease in T0 and T1 plants leads to somatic mutations and 235

consequently to chimeric plants To overcome the potential problems associated with 236

stable integration of DNA vectors containing Cas9-gRNA components gRNA in the 237

form of in vitro synthesized RNA molecules was co-bombarded with the Cas9 DNA 238

vector and analyzed for mutations by amplicon sequencing 7 days post-transformation 239

As shown in Table 4 in contrast to the delivery of both Cas9 and gRNA expressed from 240

DNA vectors the mutation frequencies observed when LIG-CR3 gRNA delivered as 241

RNA were approximately 100-fold lower (26 vs 002 respectively) 242

One possible explanation for this difference may be the requirement for 243

coincident function of Cas9 and gRNA was not met when gRNA was delivered as RNA 244

and Cas9 as a DNA vector To test this idea two Agrobacterium vectors (Supplemental 245

Fig S2) containing maize-codon optimized Cas9 under the transcriptional control of a 246



12

constitutive (maize UBI) or a temperature regulated (maize MDH) promoter were 247

introduced into Hi-II embryo cells to establish lines containing pre-integrated genomic 248

copies of Cas9 endonuclease These Agrobacterium T-DNAs also contained an 249

embryo-preferred END2 promoter regulating the expression of a blue-fluorescence 250

gene (AmCYAN) as a visible marker and an interrupted copy of the DsRED gene 251

transcriptionally regulated by a maize Histone 2B promoter Part of the DsRED 252

sequence was duplicated in a direct orientation (369 bp fragment) and consisted of two 253

fragments of the DsRED (RF-FP) gene which were separated by a 347 bp spacer that 254

could be targeted by gRNAs (Supplemental data) DSBs within the spacer region 255

promote intramolecular recombination restoring function to the disrupted DsRED gene 256

which results in red fluorescing cells Maize plants with single-copy T-DNA inserts 257

containing either UBICas9 or MDHCas9 were used as a source of immature embryos 258

for delivery of gRNAs as DNA expression cassettes or as described later as in vitro 259

transcribed RNA Blue-fluorescing embryos containing pre-integrated Cas9 were 260

excised and incubated at 280C (UBICas9) or at 370C (MDHCas9) for 24 hours Post-261

bombardment embryos with MDHCas9 were incubated at 370C for 24 hours and then 262

moved to 280C As shown in Supplemental Figure S3 in contrast to control (no gRNA) 263

UBICas9 and MDHCas9 containing embryos bombarded with two DNA-expressed 264

gRNAs that targeted sequences within the 347 bp spacer readily produced red 265

fluorescing foci after 5 days demonstrating expression of functional Cas9 protein in 266

these plants 267

To measure mutation frequencies at the LIG and MS26 endogenous target sites 268

LIG-CR3 and MS26-CR2 gRNAs as DNA vectors (25 ngshot) or as RNA molecules 269

(100 ngshot) were delivered into MDHCas9 and UBICas9 containing embryo cells 270

with temperature treatments described above In these experiments embryos were 271

harvested two days post-bombardment and analyzed by amplicon sequencing As 272

opposed to the results observed in the previous experiment nearly similar frequencies 273

were detected for gRNAs delivered as DNA vectors and as RNA molecules particularly 274

in the case of Cas9 regulated by the Ubiquitin promoter (Table 4) Together these data 275

demonstrate that delivery of gRNA in the form of RNA directly into maize cells 276



13

containing a pre-integrated Cas9 is a viable alternative to DNA delivery for the 277

generation of mutations in plant cells 278

279

Editing Maize ALS2 280

Sulfonylurea herbicides prevent branched amino acid biosynthesis in plants due 281

to the inhibition of the enzyme acetolactate synthase (ALS) (Haughn et al 1988 Lee et 282

al 1988 Yu and Powles 2014) Resistance to one of these herbicides chlorsulfuron 283

has been described as a result of single amino acid changes in the ALS protein at 284

position 197 in Arabidopsis (Pro to Ser) (Haughn et al 1988) and tobacco (Pro to Gln 285

or Ala) (Lee et al 1988) and at a corresponding location position 178 in soybean (Pro 286

to Ser) (Walter et al 2014) 287

ALS was chosen for gene editing to demonstrate that RNA-guided Cas9 can 288

facilitate specific DNA sequence changes in a native maize gene There are two ALS 289

genes in maize ALS1 and ALS2 located on chromosomes 4 and 5 respectively (Burr 290

and Burr 1991) with 94 sequence identity at the DNA level In the first experiments 291

gRNA-expressing DNA vectors targeting three different sites within both ALS1 and 292

ALS2 genes were tested (Fig 1D and Supplemental Table S1) As these gRNAs were 293

not gene-specific both ALS1 and ALS2 were mutated with approximately the same 294

efficiency (data not shown) and resulted in low stable event recovery As acetolactate 295

synthase is a critical enzyme for cell function in plants callus events containing 296

simultaneous bi-allelic knockouts of ALS1 and ALS2 genes would not be expected to 297

survive It is likely that the rare events recovered in this experiment did not receive 298

gRNA or Cas9 necessary to generate mutations and therefore contained one or more 299

functional ALS alleles Alternatively alleles may have been mutated but still retained 300

wild-type function To overcome this problem an ALS2 specific ALS-CR4 gRNA (Fig 301

2A) was designed based on the polymorphisms between ALS1 and ALS2 nucleotide 302

sequences and tested As shown in Figure 2B amplicon sequence analysis revealed 303

that ALS2 was highly preferred over ALS1 when ALS-CR4 gRNA was used One 304

important difference contributing to this result was that ALS1 lacks an NGG PAM 305

sequence required for DNA cleavage by the S pyogenes Cas9 endonuclease 306



14

To generate ALS2 edited alleles a 794 bp fragment of homology was cloned into 307

a plasmid vector and two 127 nt single-stranded DNA oligos were tested as repair 308

templates (Fig 2C) The 794 bp fragment had the same sequence modifications as 309

Oligo1 Repair templates contained several nucleotide changes in comparison to the 310

native sequence (Fig 2C and Supplemental data) Single-stranded Oligo1 and the 794 311

bp repair templates included a single nucleotide change which would direct editing of 312

DNA sequences corresponding to the proline at amino acid position 165 to a serine 313

(P165S) as well as three additional changes within the ALS-CR4 target site and PAM 314

sequence Modification of the PAM sequence within the repair template altered the 315

methionine codon (AUG) to isoleucine (AUU) which naturally occurs in the ALS1 gene 316

(Fig 2 A and C) A second 127 nt single-stranded oligo repair template (Oligo2) was 317

also tested which preserved the methionine at position 157 but contained three 318



15

additional single nucleotide changes in the sequence which would influence base 319

pairing with the ALS-CR4 gRNA (Fig 2C) 320

Approximately 1000 immature embryos per treatment were bombarded with the 321

two oligo or single plasmid repair templates Cas9 ALS-CR4 gRNA and MoPAT-322

DsRED in DNA expression cassettes and placed on media to select for bialaphos 323

resistance conferred by PAT Five weeks post-transformation two hundred (per 324

treatment) randomly selected independent young callus sectors growing on selective 325

media were separated from the embryos and transferred to fresh bialaphos plates The 326

remaining embryos (gt800 per treatment) with developing callus events were transferred 327

to the plates containing 100 ppm of chlorosulfuron as direct selection for an edited ALS2 328

gene A month later a total of 384 randomly picked callus sectors growing on bialaphos 329

(approximately 130 events for each repair template) and 7 callus sectors that continued 330

growing on media with chlorsulfuron were analyzed by PCR amplification and 331

sequencing Edited ALS2 alleles were detected in nine callus sectors two derived from 332

the callus sectors growing on bialaphos and generated using the 794 bp repair DNA 333

template and the remaining 7 derived from chlorosulfuron resistant callus sectors 334

edited using the 127 nt single-stranded oligos three by Oligo1 and four by Oligo2 (Fig 335

2D and Supplemental Table S4) The second ALS2 allele in these callus sectors was 336

mutated as a result of NHEJ repair (data not shown) Analysis of the ALS1 gene 337

revealed only wild-type sequence confirming high specificity of the ALS-CR4 gRNA 338

Plants were regenerated from 7 out of 9 callus sectors containing edited ALS2 339

alleles for additional molecular analysis and progeny testing DNA sequence analysis of 340

ALS2 alleles confirmed the presence of the P165S modifications as well as the other 341

nucleotide changes associated with the respective repair templates T1 and T2 progeny 342

of two T0 plants generated from different callus events (794 bp repair DNA and Oligo2) 343

were analyzed to evaluate the inheritance of the edited ALS2 alleles Progeny plants 344

derived from crosses using pollen from wild type Hi-II plants were analyzed by 345

sequencing and demonstrated sexual transmission of the edited alleles observed in the 346

parent plant with expected 11 segregation ratio (5756 and 4749 respectively) To test 347

whether the edited ALS sequence confers herbicide resistance selected four-week old 348

segregating T1 plants with edited and wild-type ALS2 alleles were sprayed with four 349



16

different concentrations of chlorsulfuron (50 100 (1x) 200 and 400 mgliter) Three 350

weeks after treatment plants with an edited allele showed normal phenotype while 351

plants with only wild-type alleles demonstrated strong signs of senescence (Fig 3A) In 352

addition embryos isolated from seed that had been pollinated with wild-type HI-II pollen 353

were germinated on media with 100 ppm of chlorsulfuron Fourteen days after 354

germination plants with edited alleles showed normal height and a well-developed root 355

system while plants with wild-type alleles were short and did not develop roots (Fig 356

3B) 357

These experiments demonstrate that Cas9-gRNA can stimulate HDR-dependent 358

targeted sequence modifications in maize resulting in plants with an edited endogenous 359

gene which properly transmits to subsequent generations 360

361

HDR-Mediated Gene Insertion 362



17

In contrast to Agrobacterium- or biolistic-mediated delivery of trait genes which can 363

often result in random insertions and potential disruption of native gene function 364

double-strand breaks using site-directed nucleases would allow for precise gene 365

integration at a desired genomic location In this experiment given the differences in 366

mutation frequencies observed between RNA-guided Cas9 and meganucleases 367

described above the ability of these two DSB technologies to promote site-specific 368

gene insertion was compared in maize As LIG-CR3-guided Cas9 cleaves genomic 369

DNA at the same position as LIG34 meganuclease these DSB reagents were selected 370

for this study DNA donor repair template contained the constitutively expressed PAT 371

gene (UBIMoPAT) flanked by approximately 10 kb of DNA fragments homologous to 372

genomic sequences immediately adjacent to the LIG cleavage site (Supplemental Fig 373

S1 S4 and Supplemental data) This donor vector was co-bombarded with Cas9 and 374

LIG-CR3 gRNA expression cassettes or LIG34 homing endonuclease vector In a 375

separate experiment Cas9 gRNA and a modified donor DNA template containing UBI 376

regulated MoPAT-DsRED fusion flanked with the same regions of homology were 377

delivered on the same vector (Supplemental Fig S4) To compare different delivery 378

systems this vector was moved into Agrobacterium strain LBA4044 and used in an 379

Agrobacterium-meditated transformation experiment 380

As delivery of the donor DNA would result in both random and targeted 381

insertions approximately two month old callus sectors growing on bialaphos-containing 382

media were screened for mutations and gene insertions at the LIG target site In 383

contrast to sectors derived from biolistic-transformed LIG34 meganuclease qPCR 384

copy-number analysis revealed that Cas9-gRNA yielded a higher target site mutation 385

frequency (gt83 vs 6 mutant reads Table 5) Notably for the Cas9-gRNA system 386

the mutation frequency was independent of the delivery method (bombardment or 387

Agrobacterium-mediated transformation) Callus events that contained site-specific 388

gene insertions were identified by junction PCR using primer pairs shown in Figure 4A 389

Amplification products from positive integration events were verified by subcloning and 390

sequencing As shown in Table 5 two of the 288 callus events in the meganuclease 391

experiment yielded amplicons across both junctions (07 HDR frequency) while 392

biolistic delivery of Cas9-gRNA and donor DNA resulted in frequencies of 25 and 4 393



18

when used either separately or as a single vector respectively Screening of the 192 394

callus events from Agrobacterium-mediated delivery did not produce amplicons 395

indicative of HDR-mediated gene insertions (Table 5) suggesting that biolistic 396

transformation is the preferred delivery method to generate gene insertions at the LIG 397

site in maize 398

T0 plants regenerated from 7 independent HDR positive events generated in the 399

experiment when donor DNA Cas9 and gRNA were delivered separately were used 400

for additional molecular characterization DNA blot hybridization analysis of 401

regenerated plants was conducted using genomic- and donor DNA-specific probes 402

shown in Figure 4A These analyses in addition to sequencing across the entire 403

inserted fragment (Supplemental data) demonstrated that plants regenerated from two 404

events (Events 1 and 2) contained a single copy of an intact MoPAT gene indicative of 405

homology-directed gene insertion at the LIG target site (Fig 4) Plants regenerated 406



19

from the remaining 5 events contained extra rearranged and randomly integrated 407

copies of MoPAT It should be noted that upon further molecular examination of Events 408

1 and 2 by PCR analysis the Cas9 and gRNA DNA vectors were not detected in plants 409

suggesting that transient expression of these components was sufficient to stimulate 410

gene integration 411

To examine transmission and segregation of MoPAT T0 plants from Events 1 412

and 2 were fertilized with wild-type pollen to produce T1 seeds Ninety-six T1 progeny 413

plants from Event 1 and 64 plants from Event 2 were analyzed by junction PCR and gel 414

electrophoresis and demonstrated expected Mendelian segregation of the gene 415

integrated at the LIG target site (4848 and 3133 respectively) DNA hybridization 416

analysis performed on 20 random junction PCR positive T1 plants from both events 417

using DNA probes described above confirmed nuclear stability and integrity of MoPAT 418

in progeny plants (data not shown) 419

Together these genetic and molecular data demonstrate that maize optimized 420

RNA-guided Cas9 can be used to stimulate homology-directed repair for the insertion of 421

genes at specific locations in maize chromosomes Moreover in these experiments the 422

frequency of site-specific trait integration depended upon the endonuclease target site 423

cleavage efficiency and repair template delivery method 424

425

426



20

DISCUSSION 427

During the past decade several technologies ZFNs meganucleases TALENs 428

and recently Cas9-gRNA system were developed to generate targeted DSB that can 429

be used for genome editing by NHEJ or HDR DSB technologies have three major 430

applications in plant biotechnology gene mutagenesis resulting in gene knock-outs 431

gene editing allowing modification of a gene product or metabolic pathway in a desired 432

way and site-specific trait insertion which would allow gene stacking in plant breeding 433

programs Here we reported successful application of the Cas9-gRNA technology to 434

generate multiple gene knock-outs ALS2 gene editing resulting in plants resistant to 435

chlorsulfuron and site-specific gene integration in an important crop species maize 436

437

Guide RNA Evaluation by Amplicon Sequencing 438

Maize transformation is a time consuming process often taking three to four 439

months from DNA delivery into immature embryo cells to plant regeneration Thus 440

testing the DSB reagents is an important step in developing proper tools for genome 441

editing experiments The rapid method based on amplicon sequencing described here 442

allows for evaluation of mutation frequencies at any chromosomal site targeted by 443

different gRNAs within two weeks after transformation Analysis of multiple gRNAs 444

(including ones not presented in this report) indicated that gt90 of the guides tested led 445

to a high frequency of mutations at the corresponding target sites Additional studies 446

will be needed to determine if the inability of certain guide RNAs that did not promote 447

high frequencies of mutations in planta can be attributed to such factors as target site 448

accessibility secondary structure of gRNA molecules andor high frequency of flawless 449

DSB repair at some target sites 450

451

NHEJ-Mediated Gene Mutagenesis 452

Selected gRNAs with high mutation frequencies were used in multiplexed 453

(duplexed and triplexed) targeted mutagenesis experiments All genes were targeted 454

with 100 success rate and more than 80 of the regenerated T0 plants contained 455

biallelic mutations These results along with previous publications illustrate that the 456

Cas9-gRNA technology can be used to efficiently promote the alteration of individual or 457



21

multiple gene sequences (for review see Kumar and Jain 2015) gene families or 458

homologous genes in polyploid species (Wang et al 2014) and delete chromosomal 459

segments of various sizes (Brooks et al 2014 Zhou et al 2014 Gao et al 2015) In 460

this report analysis of T0 plants and T1 segregating populations indicated that stably 461

integrated Cas9 and gRNA expression cassettes remain active often resulting in 462

chimeric T0 plants and segregation distortions Similar observations have been 463

previously reported in several plant species (Brooks et al 2014 Feng et al 2014 464

Jiang et al 2014 Wang et al 2014 Zhang et al 2014) At the same time T0 plants 465

regenerated from callus events without integrated Cas9-gRNA components were not 466

chimeric and exhibited proper sexual transmission of the mutated alleles to the next 467

generation These results suggest that transient delivery of Cas9 and gRNA is sufficient 468

to mutagenize multiple alleles and can speed the discovery and breeding process In 469

mammalian systems both Cas9 and gRNA can be delivered transiently in the form of 470

RNA-protein complex providing high mutation frequencies (Kim et al 2014 Lin et al 471

2014 Zuris et al 2015) However this approach has limited application among plant 472

species due to the currently available transformation methods Therefore one method 473

to limit the Cas9-gRNA system to transient activity is to deliver gRNA in the form of RNA 474

molecules rather than DNA expression cassettes In this report simultaneous delivery 475

of gRNA in the form of RNA and a DNA vector containing a Cas9 expression cassette 476

yielded about 100-fold lower frequencies of mutations in comparison to the control in 477

which both components were delivered as DNA vectors Because Cas9-gRNA is a two 478

component system both gRNA and Cas9 protein must be present in a cell at the same 479

time In the experiment in which gRNA and a Cas9 gene were delivered 480

simultaneously this condition likely was not met due to the predicted rapid turnover of 481

RNA molecules and the delayed and therefore non-coincidental expression of 482

functional Cas9 protein encoded on a DNA vector To overcome this challenge maize 483

lines containing constitutively or conditionally expressed pre-integrated Cas9 were 484

generated Amplicon sequence analysis performed on embryos two days after 485

transformation demonstrated that transient gRNA delivery into the maize embryo cells 486

with pre-integrated Cas9 resulted in mutation frequencies comparable to delivery of 487

Cas9 and gRNA as DNA vectors It is likely that the frequency of mutations would be 488



22

reduced in comparison to the experiments when DNA vectors were used to deliver 489

gRNAs because expression cassettes often stably integrate into the genome and as 490

the result the Cas9-gRNA system continues to be active Nevertheless delivery of 491

gRNA as RNA may not limit successful gene editing and site-specific gene integration 492

as gene insertion events were recovered without stable integration of either Cas9 or 493

gRNA expression vectors Therefore gRNA delivery in the form of RNA can be a viable 494

alternative to gRNA delivery in DNA vectors for plant gene and genome editing 495

496

HDR-Mediated Gene Editing and Targeted Gene Insertion 497

While gene mutagenesis can be the result of the error-prone process of DNA 498

DSB repair through NHEJ gene editing and targeted gene integration rely on the HDR 499

repair pathway Cas9-gRNA has been well documented in different plant species for 500

targeted gene mutagenesis both single and multiplexed (for review see Kumar and 501

Jain 2015) however examples of HDR-mediated gene editing and site-specific gene 502

insertion have been limited to model systems (Li et al 2013 Shan et al 2013) Until 503

now delivery of the gene of interest and its targeted insertion via HDR in plants remains 504

a serious challenge (Feng et al 2013 Voytas and Gao 2014) 505

Genes and genomes in many important crop species are often present in 506

multiple copies due to rearrangements duplications andor polyploidization In some 507

instances all copies of a gene may need to be mutagenized to result in complete 508

inactivation However in some cases only one copy of a gene family would require 509

editing to produce a desired phenotype In the present study the acetolactate synthase 510

(ALS) gene was chosen to test the ability of the Cas9-gRNA system to facilitate 511

endogenous gene editing via HDR in maize immature embryo cells transformed by 512

particle bombardment ALS is a well characterized gene family consisting of two genes 513

ALS1 and ALS2 which can be edited to confer plant resistance to sulfonylurea class 514

herbicides Several gRNAs targeting both genes simultaneously or ALS2 uniquely 515

were tested Delivery of a guideRNA targeting both ALS1 and ALS2 lead to low 516

recovery of stable events likely due to the high frequency of biallelic knock-out 517

mutations and the important nature of the gene However when an ALS2-specific 518

gRNA was tested typical frequencies of event recovery were observed To edit ALS2 519



23

three different repair templates (double-stranded vector DNA and two single-stranded 520

oligos) were tested All three experimental designs resulted in editing of ALS2 521

indicating that small single-stranded DNA oligonucleotides were sufficient for gene 522

editing experiments in maize Although all regenerated plants contained integrated and 523

presumably functional copies of Cas9 and gRNA expression cassettes only wild-type 524

alleles of ALS1 were observed suggesting high targeting specificity by the ALS2-CR4 525

gRNA This experiment demonstrated that by carefully choosing target sites and 526

corresponding gRNAs multiple or individual genes within a family may be targeted if 527

sufficient nucleotide polymorphisms are present between the family members 528

Targeted gene insertion allows clustering of multiple genes in a genomic location 529

which would allow for segregation as a single locus significantly simplifying the 530

breeding process Previously gene insertion at a specific target site was demonstrated 531

using ZFNs in tobacco (Cai et al 2009) and maize (Shukla et al 2009 Ainley et al 532

2013) and meganucleases in cotton (DHalluin et al 2013) The Cas9-gRNA system 533

was tested for its ability to facilitate targeted gene insertion in maize immature embryo 534

cells in comparison to a meganuclease targeting the same genomic location Two 535

different delivery systems biolistic and Agrobacterium-mediated transformation were 536

tested Only particle bombardment experiments resulted in events with a gene inserted 537

into the target site through an HDR process Delivery of the donor DNA on the same 538

plasmid with Cas9-gRNA components yielded approximately two times higher 539

frequency of integration events in comparison to experiments in which components 540

were delivered as separate vectors In this experiment Cas9-gRNA yielded 541

approximately 5 times more integration events than LIG34 meganuclease This 542

observed difference was likely due to the higher activity of Cas9-LIG-CR-3 gRNA as 543

determined by the mutation frequencies measured at the target site The inability to 544

recovery integration events in the Agrobacterium-mediated experiment may be 545

explained by the low number of copies of the T-DNA delivered into the plant cell 546

compared with particle bombardment which delivers multiple copies of all DNA 547

molecules However this does not necessarily mean that Agrobacterium transformation 548

cannot result in HDR-mediated gene integration but likely indicates lower frequency of 549

such events (Svitashev unpublished observations) Therefore selection of a delivery 550



24

method is very important and should be considered based on the goal of individual 551

experiments 552

The efficiency of gene editing and targeted gene insertions has typically been 553

associated with the activity of a nuclease and therefore frequency of mutations at a 554

given target site Although the ability of a nuclease to generate DSB is the most 555

important condition this is not the only requirement and can sometimes be misleading 556

in predicting frequency of HDR at the target site Factors such as efficiency of DSB 557

repair target site accessibility presence of repetitive sequences close to the target site 558

and yet to be discovered factors may significantly affect frequencies of gene editing and 559

targeted gene insertions Our unpublished data indicate that although similar mutation 560

rates can be observed at different target sites gene editing and gene insertion 561

frequencies can vary from less than 1 to as high as 15 562

563

CONCLUSIONS 564

Only three years after the first publications introduced the Cas-gRNA system it 565

has become the DSB technology of choice for many laboratories working in the field of 566

genome editing The advantages of the system lay not only in its simplicity and activity 567

but also in its versatility to more fully address problems associated with DSB technology 568

applications in plant biology These challenges include the inability to generate DSBs at 569

a specific genomic location and lack of complete specificity that can result in off-target 570

DNA cleavage In the not too distant future increasing the density of target sites may 571

be achieved by the discovery and utilization of new Cas9 proteins with different PAM 572

recognition sequences PAM diversity would be particularly useful in plant species with 573

high AT content while longer PAM sequences may increase the proportion of unique 574

and highly specific target sites Testing of S pyogenes Cas9-gRNA in a variety of plant 575

species has resulted in extremely low frequency of non-specific DNA cleavage and 576

therefore high specificity of the system (Feng et al 2013 Shan et al 2013 Brooks et 577

al 2014 Zhang et al 2014) While targeting various sites may yield different results 578

incorporation of the Cas9 D10A nickase (Gasiunas et al 2012) may further improve 579

gene editing options for Cas9 as the nickase version has been successfully applied to 580



25

improve specificity (Mali et al 2013 Cho et al 2014 Fauser et al 2014) 581

Furthermore transient delivery of purified Cas9 protein and gRNA (as a 582

ribonucleoprotein complex) into human culture cells has demonstrated high frequencies 583

of targeted mutagenesis and gene editing while reducing off-target mutations (Kim et 584

al 2014 Lin et al 2014) These combined approaches may further improve the 585

precision and accuracy of genome modification in plants Thus targeted mutagenesis 586

or editing of specific genes or gene families to yield new allelic variants will no longer be 587

a limiting step for the discovery of gene function and establishment of their relationship 588

to complex biological pathways Adoption of robust genome modification methods to 589

introduce genetic variation aided by inexpensive sequencing and new plant 590

transformation methods will accelerate the continued development of new breeding 591

techniques to address challenges of modern agriculture 592

593

MATERIALS AND METHODS 594

595

Plasmids and Reagents Used for Plant Transformation 596

Standard DNA techniques as described in Sambrook et al (Sambrook et al 597

1898) were used for vector construction The Cas9 sequence from Streptococcus 598

pyogenes M1 GAS (SF370) was maize codon-optimized and designed to contain the 599

potato ST-LS1 intron (Accession number X04753 (nucleotides 2837-2892) at 600

nucleotide position (+402-591) To facilitate nuclear localization of the Cas9 protein in 601

maize cells the SV40 (MAPKKKRKV) and the A tumefaciens VirD2 602

(KRPRDRHDGELGGRKRAR) nuclear localization signals were added at the amino and 603

carboxyl-termini of the Cas9 open reading frame respectively (Supplemental data) 604

The chemically synthesized Cas9 gene contained on a 4335 kb NcoI-HpaI DNA 605

fragment was subcloned into PHP17720 (Sabelli et al 2009) digested with NcoI-HpaI 606

which links Cas9 to a maize constitutive Ubiquitin-1 promoter including the first intron (ndash607

899 to+1092 Christensen et al 1992) while transcription is terminated by the addition 608

of the 3rsquo sequences from the potato proteinase inhibitor II gene (PinII) (nucleotides 2ndash609

310 (An et al 1989) to generate UBICas9 vector Single guide RNAs (gRNAs) were 610



26

designed using the methods described by Mali et al 2013 A maize U6 polymerase III 611

promoter and terminator residing on chromosome 8 (nt 165535024-165536023 Maize 612

(B73) Public Genome Assembly (AGP_v38)) were isolated and used to direct initiation 613

and termination of gRNAs respectively Two BbsI restriction endonuclease sites were 614

introduced in an inverted tandem orientation with cleavage orientated in an outward 615

direction as described in Cong et al 2013 to facilitate the rapid introduction of maize 616

genomic DNA target sequences into the gRNA expression constructs (Supplemental 617

data) Only target sequences starting with a G nucleotide were used to promote 618

favorable polymerase III expression of the gRNA The guideRNA expression cassettes 619

contained on a 11 kb XhoI-EcoRI DNA fragment were subcloned into Bluescript SK 620

vectors and used for bombardment and subsequent vector construction 621

Two Agrobacterium-transformation vectors were designed and introduced into 622

maize to generate stably integrated Cas9 expression cassettes To generate 623

constitutively expressed Cas9 the UBICas9 vector was digested with HindIII-EcoRI to 624

release a 66 kb HindIII-EcoRI fragment (UBICas9) and this fragment was subcloned 625

into HindIII-EcoRI digested plasmid pSB11 (Ishida et al 1996) To generate the 626

UBICas9 T-DNA vector the pSB11-UbiCas9 vector was digested with NotI and a 58 627

kb NotI fragment containing the blue-fluorescence gene (CFP AmCYAN1 Clontech 628

Laboratories Inc CA USA) regulated by the maize END2 promoter (Chromosome 8 nt 629

171120325-171119383 Maize (B73) Public Genome Assembly (AGP_v38)) a visible 630

marker for the identification of transformed embryos) and a red fluorescence gene 631

(pDsRED Express Product number 632412 Clontech Laboratories Inc CA USA) 632

regulated by the maize Histone 2B promoter (Chromosome 3 nt 16569805ndash16571259 633

Maize (B73) Public Genome Assembly (AGP_v38)) was subcloned downstream of 634

UBICas9 A portion of DsRED gene in this vector was duplicated (nt 148-516) in a 635

direct orientation with 369 bp of overlap The duplicated fragments were separated by a 636

347-bp spacer contained sequences compatible for recognition and targeting by gRNAs 637

and the LIG34 meganuclease (Supplemental Fig S2) To generate a conditionally 638

expressed Cas9 (MDHCas9) a 1048 bp fragment of temperature regulated promoter 639

from the maize mannitol dehydrogenase gene (MDH Chromosome 2 nt 8783228-640

8784275 Maize (B73) Public Genome Assembly (AGP_v32)) was modified to contain 641



27

an RcaI restriction site at the 3rsquo end of the sequence (Cigan and Unger-Wallace 2013) 642

to accommodate translational fusion to the maize optimized Cas9 to generate 643

MDHCas9 expression cassette The 66 kb HindIII-EcoRI DNA fragment in UBICas9 644

T-DNA vector was replaced with the MDHCas9 DNA contained on a 57 kb HindIII-645

EcoRI DNA fragment to generate the MDHCas9 T-DNA vector These plasmids were 646

introduced into Agrobacterium strain LBA4404 (Komari et al 1996) by electroporation 647

using a Gene Pulser II (Bio-Rad CA USA) as described by Gao (Gao et al 2010) 648

To generate gRNA in the form of RNA molecules the maize-optimized U6 649

polymerase III gRNA expression cassettes were amplified by PCR using a 5rsquo 650

oligonucleotide primer that also contained the sequence of the T7 polymerase promoter 651

and transcriptional initiation signal just 5rsquo of the spacer to gene T7 in vitro transcription 652

was carried out with the AmpliScribetrade T7-Flashtrade kit (Epicentre WI USA) according 653

to the manufacturerrsquos recommendations and products were purified using NucAwaytrade 654

Spin Columns (Invitrogen Life Technologies Inc USA) followed by ethanol 655

precipitation 656

The LIG34 and the Ems26 endonucleases vectors were previously described 657

(Gao et al 2010 Djukanovic et al 2013 respectively) Plasmids containing cell 658

division promoting polypeptides ZmODP2 and ZmWus and selectable and visible 659

markers MoPAT-DsRED (a translational fusion of the bialophos resistance gene 660

phosphinothricin-N-acetyl-transferase and the red fluorescent protein DsRED) were 661

previously described in Ananiev et al 2009 662

For ALS2 gene editing single-stranded 127 nt long oligos (sense and antisense 663

strands) homologous to the fragment spanning the ALSCas-4 target site with 4 and 7 664

single nucleotide changes (Fig 2C) were synthesized (Integrated DNA Technologies 665

IA USA) SNPs within gRNA target site and PAM sequence were introduced to prevent 666

the donor DNA from cleavage by Cas9 protein and SNPs a C to T Oligo1 and a C to T 667

and a G to C replacements downstream from the cleavage site to change proline to 668

serine at position 165 in the ALS2 gene These oligos (sense and antisense strands) 669

were annealed to generate double-stranded DNA digested with BcoDI and ligated to 670

PCR amplified genomic DNA fragments upstream and downstream from the oligo 671

sequence with primers 5rsquo-ACAGCCGCCGCAACCATGGCCA-3rsquo (forward) and 5rsquo-672



28

ACCATGGGGACGGAATCGAGCA-3rsquo (reverse) and 5rsquo-673

ATAAGAGTAGTTGTGACCGGGA-3rsquo (forward) and 5rsquo-674

CTGCTCAAGCAACTCAGTCGCAGGG-3rsquo (reverse) respectively also digested with 675

BcoDI to extend the total homology region to 794 bp and cloned into the pUC19 vector 676

Vectors for HDR-mediated targeted genome integration were generated by 677

flanking a UBIMoPAT or UBIMoPAT-DsRED fusion with 1099 bp and 1035 bp maize 678

genomic DNA fragments homologous to the sequences immediately upstream and 679

downstream from the cleavage site of the of LigCas-3 target HR1 and HR2 680

respectively (Fig 4A) Vector containing the MoPAT-DsRED fusion was retrofitted 681

either with UBICas9 and U6gRNA or LIG34 meganuclease expression cassettes 682

(Supplemental Fig S4B) For Agrobacterium-mediated transformation vector 683

containing expression cassettes shown on Supplemental Figure S4B was moved into 684

the A tumefaciens strain LBA4404 by electroporation using a Gene Pulser II (Bio-Red 685

CA USA) 686

687

Plant Material 688

Publicly available maize (Zea mays L) hybrid high type II (Hi-II) line (Armstrong 689

and Green 1985) was obtained from internal Pioneer sources 690

To generate plants with either constitutive or regulated expression of Cas9 691

protein constructs described above and shown in Supplemental Figure S2 were used to 692

transform Hi-II maize embryos using Agrobacterium-mediated transformation 693

Regenerated T0 plants were screened by qPCR and DNA blot hybridization analysis for 694

single-copy T-DNA insertions Independent UBICas9 (4) and DHCas9 (11) T-DNA 695

containing plants were identified T0 plants were fertilized with wild-type Hi-II pollen and 696

blue fluorescing embryos were used for further biolistic transformation experiments 697

698

Maize Transformation 699

Biolistic-mediated transformation of maize immature embryos was performed as 700

described in Ananiev et al (Ananiev et al 2009) with slight modification Briefly the 701

DNA was precipitated onto 06 microm (average diameter) gold microprojectiles using a 702

water-soluble cationic lipid TransITreg-2020 (Mirus WI USA) as follows 50 microl of gold 703



29

particles (water solution 10 mgml) and 1 microl of TransIT-2020 water solution were added 704

to the premixed DNA constructs (DNA and RNA amounts used in each experiment are 705

shown in Supplemental Table S5) mixed gently and incubated on ice for 10 min DNA-706

coated gold particles were then centrifuged at 10000 rpm for 1 minute The pellet was 707

rinsed with 100 microl of absolute alcohol and resuspended by a brief sonication 708

Immediately after sonication DNA-coated gold particles were loaded onto the center of 709

a macrocarrier (10 microl each) and allowed to air dry Immature maize embryos 8-10 days 710

after pollination were bombarded using a PDS-1000He Gun (Bio-Rad CA USA) with a 711

rupture pressure of 425 PSI Post-bombardment culture selection and plant 712

regeneration were performed as previously described (Gordon-Kamm et al 2002) 713

Agrobacterium-mediated transformation followed the detailed protocol previously 714

described in Gao et al 2010 Regenerated plantlets were moved to soil where they 715

were sampled and grown to maturity in greenhouse greenhouse conditions 716

717

PCR qPCR Analysis and Amplicon Sequencing 718

For PCR analysis DNA was extracted from a small amount (05 cm in diameter) 719

of callus tissue or two leaf punches as described in Gao et al 2010 PCR was 720

performed using REDExtract-N-AmpTM PCR ReadyMix (Sigma USA) or Phusionreg High 721

Fidelity PCR Master Mix (NEB MA USA) according to the manufacturerrsquos 722

recommendations PCR amplified fragments were cloned using the pCR21-TOPO 723

cloning vector (Life Technologies Inc USA) The primers used in PCR and qPCR 724

reactions are shown in Supplemental Table 3 qPCR analysis was performed as in (Wu 725

et al 2014) As mutations within a population of individual callus sectors may differ 726

from simple (single base pair insertions or deletions) to complex (deletions and 727

additions) sequence modifications target site copy-number as determined by qPCR 728

analysis was used to as a semi-qualitative method to determine mutation frequencies 729

In general mutation frequency was measured based on target site allele copy-number 730

both alleles are defined to be intact if target site copy-number is 2 and the event would 731

be called wild type Events for which qPCR revealed target site copy-number reduced 732

to one would be scored as one mutated allele and one wild type allele Finally if both 733



30

alleles of the target site are modified the copy-number is zero and event would be 734

called ldquonullrdquo 735

For deep sequencing analysis 60-90 Hi-II maize immature embryos were co-736

bombarded with the maize optimized Cas9 endonuclease and gRNA expression 737

cassettes or I-CreI modified endonuclease vectors and screenable marker UBIMoPAT-738

DsRED and cell division promoting genes UBIZmODP2 and IN2ZmWUS2 Seven 739

days after bombardment 20-30 of the most uniformly transformed embryos (based on 740

transient expression of DsRED) from each treatment were pooled and total genomic 741

DNA was extracted The DNA region surrounding the intended target site was amplified 742

by PCR using Phusionreg High Fidelity PCR Master Mix (NEB MA USA) adding the 743

amplicon-specific barcodes and Illumina sequencing through two rounds of PCR The 744

primers used in the primary PCR reaction are shown in Supplemental Table 3 and the 745

primers used in the secondary PCR reaction were 5rsquo-746

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACG-3rsquo (forward) and 5rsquo-747

CAAGCAGAAGACGGCATA-3rsquo (reverse) The resulting PCR amplifications were 748

purified with a Qiagen PCR purification spin column (Qiagen Germany) concentration 749

measured with a Hoechst dye-based fluorometric assay combined in an equimolar 750

ratio and single read 100 nucleotide-length amplicon sequencing was performed on 751

Illuminarsquos MiSeq Personal Sequencer with a 30-40 (vv) spike of PhiX control v3 752

(Illumina FC-110-3001) to off-set sequence bias Only those reads with a ge1 nucleotide 753

INDEL arising within the 10 nt window centered over the expected site of cleavage and 754

not found in the negative controls were classified as NHEJ mutations NHEJ mutant 755

reads with the same mutation were counted and collapsed into a single read and the top 756

10 most prevalent mutations were visually confirmed as arising within the expected site 757

of cleavage The total numbers of visually confirmed mutations were then used to 758

calculate the percentage of mutant reads based on the total number of reads of an 759

appropriate length containing a perfect match to the barcode and forward primer 760

761

DNA Isolation and Blot Hybridization Analysis 762

For DNA isolation and blot hybridization analyses maize leaf tissue (approximately 25 763

g fresh weight) from transformed and control plants was freeze-dried and ground Total 764



31

genomic DNA was extracted as described in Cigan et al 2001 Five micrograms of 765

DNA from each sample were digested overnight with HindIII and the DNA fragments 766

were separated on a 08 agarose gel run at 23 V overnight DNA was transferred to a 767

nylon membrane (Roche Germany) using standard protocol (Sambrook et al 1989) 768

and fixed to the membrane by UVirradiation of 150 mJ in a Gene Linker (Bio-Rad CA 769

USA) Membranes were pre-hybridized for 1 hour in DIG Easy Hyb (Roche Germany) 770

at 50degC Hybridization probes LIG-5rsquo (459 bp) LIG-3rsquo (456 bp) and Mo-PAT (446 bp) 771

were labeled by PCR with primers 5rsquo-AGCTTTATCCATCCATCCATCGC-3rsquo (forward) 772

and 5rsquo-AATTCTCGTACGTACAGCACGCG-3rsquo (reverse) 5rsquo-773

ACCCCTACAGCCACTTAGTCTCCG-3rsquo (forward) and 774

GCCAGAGAGATCGAGATCGATGGA-3rsquo (reverse) and 5rsquo-775

GTGTGCGACATCGTGAACCACT-3rsquo (forward) and 5rsquo-TCGAAGTCGCGCTGCCAGAA-776

3rsquo (reverse) respectively using the DIG Probe PCR Synthesis Kit (Roche Germany) 777

according to manufacturerrsquos recommendations Hybridization was performed overnight 778

at 50degC Membranes were washed twice in 2times SSC01 SDS at room temperature 779

followed by three washes in 01times SSC01 SDS for HR probe or 005times SSC005 780

SDS for Mo-PAT probe at 63degC Membranes were prepared for detection by washing 781

at room temperature in 1times Wash Buffer (DIG Wash and Block Buffer Set) for 10 782

minutes then 1times Blocking Buffer (DIG Wash and Block Buffer Set) for 60 minutes 783

followed by 1times Antibody solution (Anti-Digoxigenin-AP Fab Fragments (Roche 784

Germany) in 1times Blocking Buffer) for 60 minutes and finally three times in 1times Wash 785

Buffer for 8 minutes per wash Detection was done by placing membranes in 1times 786

Detection Buffer (DIG Wash and Block Buffer Set) for 5 minutes removing to a clean 787

tray and pipetting CDP-Star ready-to-use (Roche Germany) onto the membranes then 788

exposed to X-ray film (Kodak USA) 789

790

ACKNOWLEDGMENTS 791

Authors are thankful to Brian Lenderts Meizhu Yang Shan Zhao Wally Marsh 792

Susan Wagner and Min Zeng for technical and maize transformation support DuPont 793

Pioneer Controlled Environment group for managing plants and Genomics group for 794

DNA sequencing The authors extend special thanks to Brooke Peterson-Burch for 795



32

bioinformatics support Wayne Crismani Jeff Sander Doane Chilcoat Philippe Horvath 796

and the anonymous Plant Physiology reviewers for thoughtful comments and 797

suggestions 798

799



33

800

SUPPLEMENTAL MATERIAL 801 Table S1 Maize genomic sites targeted by Cas9-gRNA system 802

Table S2 T0 plant analysis by qPCR for mutations at maize target loci of 30 T0 plants 803 produced in the multiplexed Cas9-gRNA experiments 804

Table S3 List of primers used in the study 805

Table S4 Edited ALS2 events recovered using different templates and selection 806

Table S5 DNA and RNA amounts used in bombardment experiments (single shot) 807

Figure S1 Ten of the most prevalent mutations generated by either the Cas9-gRNA 808 system or the LIG34 homing endonuclease identified by deep sequencing of the PCR 809 amplicons across the corresponding target site 810

Figure S2 Agro vector for stable integration of the ZmUBICas9 into the maize 811

genome 812

Figure S3 Repair of the RF-FP gene via intramolecular homologous recombination 813

Figure S4 Constructs used for HDR-mediated targeted gene insertion at the liguleless-814

1 region using Cas-gRNA system and LIG34 meganuclease 815

Nucleotide sequence of maize optimized Cas9 816

Nucleotide sequence of LIG-CR3 gRNA expression cassette 817

Nucleotide sequence and description of the donor DNA in the integration 818

experiment 819

Nucleotide sequence and description of the RF-FP repair cassette 820

821

822



34

823



35

824



36

Table 1 Percentage of mutant reads at 5 different target sites 7 days post-825

transformation 826

Target DSB Reagents Total Number

of Reads

Number of Mutant Target Gene Reads

Percentage of Mutant Reads in

Target Gene

LIG (Chr 2)

LIG34 meganuclease 616536 1211 020

LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)

Ems26 meganuclease 512784 642 013

MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126

ALS1 (Chr 4) and ALS2

(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172

Controls Cas9 only 640063 1 000

LIG-CR1 gRNA only 646774 1 000



37

Table 2 Read counts and percentage of mutant reads at maize target sites in 827

multiplexed Cas9-gRNA experiments 7 days post-transformation 828

829

830

Target Site Co-Transformed gRNAs Total Number

of Reads

Number of Mutant Target Gene

Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178

LIGCas-3 MS26Cas-2 MS45Cas-2 648901 12094 186

MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162

MS26Cas-2 MS45Cas-2 LIGCas-3 471890 7311 155

MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831

Table 3 Mutation analysis at maize target sites in multiplexed experiments 832

Target Sites

T0 Plant

qPCR Results

LIG34 TS MS26 TS MS45 TS Stable

Integration

Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Cas9 gRNA

LIGCas-3

MS26Cas2

1 nullnull 1bp del 2bp del+

1 bp ins

1bp ins

(A) 19bp del - - Yes Yes

2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2

MS45Cas-2 3 hetnull - -

1bp ins

(C) WT 2bp del 65bp del Yes No

LIGCas-3

MS26Cas-2

MS45Cas-2

4 nullnullnull 1bp ins

(T)

large

mut

1bp ins

(T) 1 bp del 15bp del

large

mut Yes Yes

5 hetnullnull 1bp ins

(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No

833 Null indicates that both alleles are mutated het indicates that one alleles is mutated and the other one is 834 wild-type 835 Due to the size and nature of the mutation (likely the transformation vector insertion) it could not be 836 resolved by the methods used in the analysis 837



39

Table 4 Percentage of mutant reads at maize LIG and MS26 target sites produced in 838

transient gRNA delivery into wild-type embryos and embryos with pre-integrated Cas9 839

under constitutive (UBI) or regulated (MDH) promoters 840

841

Target Site

Hi-II Embryos Transformation

Percentage of Mutant Reads

2 days post-transformation


LIG

Wild-type Cas9 (DNA) + gRNA (DNA) - 260

Cas9 (DNA) + gRNA (RNA) - 002

UBICas9 gRNA (DNA) 122

gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -

842 In these experiments analysis was performed on embryos collected 7 days after transformation while in 843 the experiments with pre-integrated Cas9 analysis was done on embryos collected 2 days after 844 transformation The additional 5 days allows constitutively expressed Cas9 endonuclease to continue 845 DNA cleavage thus accounting for the difference in percentage of mutant reads in the DNA delivery 846 experiments 847



40

Table 5 Summary of gene integration at LIG target site 848

849

System Experiment Number of

Events Analyzed

Mutation Frequency LIG

target site

2 Junction PCR Positive Events

LIG34 Meganuclease + donor DNA

Separate vectors

Particle bombardment

288 6 2 (07)

LIG-CR3 gRNA + Cas9 + donor DNA

3 separate vectors


480 83 11 (25)


All in a single vector


336 86 14 (41)


Agrobacterium-mediated delivery 192 84 0

850

851


Parsed CitationsAinley WM Sastry-Dent L Welter ME Murray MG Zeitler B Amora R Corbin DR Miles RR Arnold NL Strange TL Simpson MACao Z Carroll C Pawelczak KS Blue R West K Rowland LM Perkins D Samuel P Dewes CM Shen L Sriram S Evans SL RebarEJ Zhang L Gregory PD Urnov FD Webb SR Petolino JF (2013) Trait stacking via targeted genome editing Plant BiotechnologyJournal 11 1126-1134

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

An G Mitra A Choi HK Costa MA An K Thornburg RW Ryan CA (1989) Functional analysis of the 3 control region of the potatowound-inducible proteinase inhibitor II gene Plant Cell 1 115-122


Ananiev E Wu C Chamberlin M Svitashev S Schwartz C Gordon-Kamm W Tingey S (2009) Artificial chromosome formation inmaize (Zea mays L) Chromosoma 118 157-177


Armstrong CL Green CE (1985) Establishment and maintenance of friable embryogenic maize callus and the involvement of L-proline Planta 164 207-214


Arnould S Chames P Perez C Lacroix E Duclert A Epinat J-C Stricher F Petit A-S Patin A Guillier S Rolland S Prieto J BlancoFJ Bravo J Montoya G Serrano L Duchateau P Pacircques F (2006) Engineering of Large Numbers of Highly Specific HomingEndonucleases that Induce Recombination on Novel DNA Targets Journal of Molecular Biology 355 443-458


Barrangou R Fremaux C Deveau H Richards M Boyaval P Moineau S Romero DA Horvath P (2007) CRISPR Provides AcquiredResistance Against Viruses in Prokaryotes Science 315 1709-1712


Boch J Scholze H Schornack S Landgraf A Hahn S Kay S Lahaye T Nickstadt A Bonas U (2009) Breaking the Code of DNABinding Specificity of TAL-Type III Effectors Science 326 1509-1512


Brooks C Nekrasov V Lippman Z Van Eck J (2014) Efficient gene editing in tomato in the first generation using the CRISPRCas9system Plant Physiol 166 1292-1297


Burr B Burr FA (1991) Recombinant inbreds for molecular mapping in maize theoretical and practical considerations Trends inGenetics 7 55-60


Cai C Doyon Y Ainley W Miller J DeKelver R Moehle E Rock J Lee Y-L Garrison R Schulenberg L Blue R Worden A Baker LFaraji F Zhang L Holmes M Rebar E Collingwood T Rubin-Wilson B Gregory P Urnov F Petolino J (2009) Targeted transgeneintegration in plant cells using designed zinc finger nucleases Plant Molecular Biology 69 699-709


Cho SW Kim S Kim Y Kweon J Kim HS Bae S Kim J-S (2014) Analysis of off-target effects of CRISPRCas-derived RNA-guidedendonucleases and nickases Genome Research 24 132-141


Choulika A Perrin A Dujon B Nicolas J (1995) Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae Mol Cell Biol 15 1968-1973


Christensen AH Sharrock RA Quail PH (1992) Maize polyubiquitin genes structure thermal perturbation of expression andhttpsplantphysiolorgDownloaded on November 7 2020 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

transcript splicing and promoter activity following transfer to protoplasts by electroporation Plant Mol Biol 18 675-689Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Cigan AM Unger-Wallace E inventors Nov 21 2013 Inducible promoter sequences for regulated expression and methods of useUS Patent Application No 20130312137 A1


Cigan AM Unger E Xu R-J Kendall TL Fox TW (2001) Phenotypic complementation of ms45 mutant maize requires tapetalexpression of the Ms45 gene Sex Plant Reprod 14 135-142


Cong L Ran FA Cox D Lin S Barretto R Habib N Hsu PD Wu X Jiang W Marraffini LA Zhang F (2013) Multiplex GenomeEngineering Using CRISPRCas Systems Science 339 819-823


Cornu TI Thibodeau-Beganny S Guhl E Alwin S Eichtinger M Joung JK Cathomen T (2007) DNA-binding Specificity Is a MajorDeterminant of the Activity and Toxicity of Zinc-finger Nucleases Mol Ther 16 352-358


DHalluin K Vanderstraeten C Van Hulle J Rosolowska J Van Den Brande I Pennewaert A DHont K Bossut M Jantz D RuiterR Broadhvest J (2013) Targeted molecular trait stacking in cotton through targeted double-strand break induction PlantBiotechnol J 11 933-941


Djukanovic V Smith J Lowe K Yang M Gao H Jones S Nicholson MG West A Lape J Bidney D Falco CS Jantz D Lyznik LA(2013) Male-sterile maize plants produced by targeted mutagenesis of the cytochrome P450-like gene (MS26) using a re-designedI-CreI homing endonuclease The Plant Journal 76 888-899


Doudna JA Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9 Science 346 1258096Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Fauser F Schiml S Puchta H (2014) Both CRISPRCas-based nucleases and nickases can be used efficiently for genomeengineering in Arabidopsis thaliana The Plant Journal 79 348-359


Feng Z Mao Y Xu N Zhang B Wei P Yang D-L Wang Z Zhang Z Zheng R Yang L Zeng L Liu X Zhu J-K (2014) Multigenerationanalysis reveals the inheritance specificity and patterns of CRISPRCas-induced gene modifications in Arabidopsis Proceedingsof the National Academy of Sciences 111 4632-4637


Feng Z Zhang B Ding W Liu X Yang D-L Wei P Cao F Zhu S Zhang F Mao Y Zhu J-K (2013) Efficient genome editing in plantsusing a CRISPRCas system Cell Res 23 1229-1232


Gao H Smith J Yang M Jones S Djukanovic V Nicholson MG West A Bidney D Falco SC Jantz D Lyznik LA (2010) Heritabletargeted mutagenesis in maize using a designed endonuclease The Plant Journal 61 176-187


Gao J Wang G Ma S Xie X Wu X Zhang X Wu Y Zhao P Xia Q (2015) CRISPRCas9-mediated targeted mutagenesis in Nicotianatabacum Plant Molecular Biology 87 99-110


Gasiunas G Barrangou R Horvath P Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage forhttpsplantphysiolorgDownloaded on November 7 2020 - Published by

Copyright (c) 2020 American Society of Plant Biologists All rights reserved

adaptive immunity in bacteria Proceedings of the National Academy of Sciences 109 E2579-E2586Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Gordon-Kamm W Dilkes BP Lowe K Hoerster G Sun X Ross M Church L Bunde C Farrell J Hill P Maddock S Snyder J SykesL Li Z Woo Y-m Bidney D Larkins BA (2002) Stimulation of the cell cycle and maize transformation by disruption of the plantretinoblastoma pathway Proceedings of the National Academy of Sciences 99 11975-11980


Halfter U Morris PC Willmitzer L (1992) Gene targeting in Arabidopsis thaliana Molecular and General Genetics MGG 231 186-193


Hanin M Volrath S Bogucki A Briker M Ward E Paszkowski J (2001) Gene targeting in Arabidopsis The Plant Journal 28 671-677Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Haughn G Smith J Mazur B Somerville C (1988) Transformation with a mutant Arabidopsis acetolactate synthase gene renderstobacco resistant to sulfonylurea herbicides Molecular and General Genetics MGG 211 266-271


Hsu Patrick D Lander Eric S Zhang F (2014) Development and Applications of CRISPR-Cas9 for Genome Engineering Cell 1571262-1278


Ishida Y Saito H Ohta S Hiei Y Komari T Kumashiro T (1996) High efficiency transformation of maize (Zea mays L) mediated byAgrobacterium tumefaciens Nat Biotechnol 14 745-750


Jacobs TB LaFayette PR Schmitz RJ Parrott WA (2015) Targeted genome modifications in soybean with CRISPRCas9 BMCbiotechnology 15 16


Jasin M Rothstein R (2013) Repair of Strand Breaks by Homologous Recombination Cold Spring Harbor Perspectives in Biology5 a012740


Jiang W Yang B Weeks DP (2014) Efficient CRISPRCas9-Mediated Gene Editing in Arabidopsis thaliana and Inheritance ofModified Genes in the T2 and T3 Generations PLoS One 9 e99225


Jinek M Chylinski K Fonfara I Hauer M Doudna JA Charpentier E (2012) A Programmable Dual-RNA-Guided DNA Endonucleasein Adaptive Bacterial Immunity Science 337 816-821


Kempin SA Liljegren SJ Block LM Rounsley SD Yanofsky MF Lam E (1997) Targeted disruption in Arabidopsis Nature 389 802-803


Kim S Kim D Cho SW Kim J Kim J-S (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purifiedCas9 ribonucleoproteins Genome Research 24 1012-1019


Komari T Hiei Y Saito Y Murai N Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higherplants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers The Plant Journal10 165-174



Kumar V Jain M (2015) The CRISPR-Cas system for plant genome editing advances and opportunities Journal of ExperimentalBotany 66 47-57


Lee KY Townsend J Tepperman J Black M Chui CF Mazur B Dunsmuir P Bedbrook J (1988) The molecular basis ofsulfonylurea herbicide resistance in tobacco The EMBO Journal 7 1241-1248


Li J-F Norville JE Aach J McCormack M Zhang D Bush J Church GM Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 Nat Biotech 31 688-691


Liang Z Zhang K Chen K Gao C (2014) Targeted Mutagenesis in Zea mays Using TALENs and the CRISPRCas System Journal ofGenetics and Genomics 41 63-68


Lin S Staahl BT Alla RK Doudna JA (2014) Enhanced homology-directed human genome engineering by controlled timing ofCRISPRCas9 delivery Elife 3 e04766


Maeder ML Thibodeau-Beganny S Osiak A Wright DA Anthony RM Eichtinger M Jiang T Foley JE Winfrey RJ Townsend JAUnger-Wallace E Sander JD Muumlller-Lerch F Fu F Pearlberg J Goumlbel C Dassie Justin P Pruett-Miller SM Porteus MH Sgroi DCIafrate AJ Dobbs D McCray Jr PB Cathomen T Voytas DF Joung JK (2008) Rapid Open-Source Engineering of CustomizedZinc-Finger Nucleases for Highly Efficient Gene Modification Molecular Cell 31 294-301


Makarova K Aravind L Wolf Y Koonin E (2011) Unification of Cas protein families and a simple scenario for the origin andevolution of CRISPR-Cas systems Biology Direct 6 38


Makarova KS Haft DH Barrangou R Brouns SJJ Charpentier E Horvath P Moineau S Mojica FJM Wolf YI Yakunin AF van derOost J Koonin EV (2011) Evolution and classification of the CRISPR-Cas systems Nat Rev Micro 9 467-477


Mali P Yang L Esvelt KM Aach J Guell M DiCarlo JE Norville JE Church GM (2013) RNA-Guided Human Genome Engineeringvia Cas9 Science 339 823-826


Miao Z-H Lam E (1995) Targeted disruption of the TGA3 locus in Arabidopsis thaliana The Plant Journal 7 359-365Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Moore JK Haber JE (1996) Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae Molecular and Cellular Biology 16 2164-2173


Offringa R Franke-van Dijk ME De Groot MJ van den Elzen PJ Hooykaas PJ (1993) Nonreciprocal homologous recombinationbetween Agrobacterium transferred DNA and a plant chromosomal locus Proceedings of the National Academy of Sciences 907346-7350


Puchta H Dujon B Hohn B (1993) Homologous recombination in plant cells is enhanced by in vivo induction of double strandbreaks into DNA by a site-specific endonuclease Nucleic Acids Research 21 5034-5040



Puchta H Dujon B Hohn B (1996) Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination Proceedings of the National Academy of Sciences 93 5055-5060


Puchta H Fauser F (2014) Synthetic nucleases for genome engineering in plants prospects for a bright future The Plant Journal78 727-741


Roth DB Wilson JH (1986) Nonhomologous recombination in mammalian cells role for short sequence homologies in the joiningreaction Molecular and Cellular Biology 6 4295-4304


Sabelli PA Hoerster G Lizarraga LE Brown SW Gordon-Kamm WJ Larkins BA (2009) Positive regulation of minichromosomemaintenance gene expression DNA replication and cell transformation by a plant retinoblastoma gene Proceedings of theNational Academy of Sciences 106 4042-4047


Sambrook J Fritsch EF Maniatis T (1989) Molecular cloning a laboratory manual Cold Spring Harbor NY Cold Spring HarborLaboratory


Shan Q Wang Y Li J Zhang Y Chen K Liang Z Zhang K Liu J Xi JJ Qiu J-L Gao C (2013) Targeted genome modification of cropplants using a CRISPR-Cas system Nat Biotech 31 686-688


Shukla VK Doyon Y Miller JC DeKelver RC Moehle EA Worden SE Mitchell JC Arnold NL Gopalan S Meng X Choi VM RockJM Wu Y-Y Katibah GE Zhifang G McCaskill D Simpson MA Blakeslee B Greenwalt SA Butler HJ Hinkley SJ Zhang L RebarEJ Gregory PD Urnov FD (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases Nature459 437-441


Smih F Rouet P Romanienko PJ Jasin M (1995) Double-strand breaks at the target locus stimulate gene targeting in embryonicstem cells Nucleic Acids Research 23 5012-5019


Smith J Bibikova M Whitby FG Reddy AR Chandrasegaran S Carroll D (2000) Requirements for double-strand cleavage bychimeric restriction enzymes with zinc finger DNA-recognition domains Nucleic Acids Research 28 3361-3369


Smith J Grizot S Arnould S Duclert A Epinat J-C Chames P Prieto J Redondo P Blanco FJ Bravo J Montoya G Pacircques FDuchateau P (2006) A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences Nucleic AcidsResearch 34 e149-e149


Szczepek M Brondani V Buchel J Serrano L Segal DJ Cathomen T (2007) Structure-based redesign of the dimerizationinterface reduces the toxicity of zinc-finger nucleases Nat Biotech 25 786-793


Terada R Johzuka-Hisatomi Y Saitoh M Asao H Iida S (2007) Gene Targeting by Homologous Recombination as aBiotechnological Tool for Rice Functional Genomics Plant Physiol 144 846-856



Terada R Urawa H Inagaki Y Tsugane K Iida S (2002) Efficient gene targeting by homologous recombination in rice 20 1030-1034Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Townsend JA Wright DA Winfrey RJ Fu F Maeder ML Joung JK Voytas DF (2009) High-frequency modification of plant genesusing engineered zinc-finger nucleases Nature 459 442-445


Voytas DF Gao C (2014) Precision Genome Engineering and Agriculture Opportunities and Regulatory Challenges PLoS Biol 12e1001877


Walter KL Strachan SD Ferry NM Albert HH Castle LA Sebastian SA (2014) Molecular and phenotypic characterization of Als1and Als2 mutations conferring tolerance to acetolactate synthase herbicides in soybean Pest Management Science 70 1831-1839


Wang Y Cheng X Shan Q Zhang Y Liu J Gao C Qiu J-L (2014) Simultaneous editing of three homoeoalleles in hexaploid breadwheat confers heritable resistance to powdery mildew Nat Biotech 32 947-951


Weber E Gruetzner R Werner S Engler C Marillonnet S (2011) Assembly of Designer TAL Effectors by Golden Gate CloningPLoS One 6 e19722


Wu E Lenderts B Glassman K Berezowska-Kaniewska M Christensen H Asmus T Zhen S Chu U Cho M-J Zhao Z-Y (2014)Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants In VitroCellular amp Developmental Biology - Plant 50 9-18


Yu Q Powles SB (2014) Resistance to AHAS inhibitor herbicides current understanding Pest Management Science 70 1340-1350Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Zhang H Zhang J Wei P Zhang B Gou F Feng Z Mao Y Yang L Zhang H Xu N Zhu J-K (2014) The CRISPRCas9 systemproduces specific and homozygous targeted gene editing in rice in one generation Plant Biotechnology Journal 12 797-807


Zhang Y Zhang F Li X Baller JA Qi Y Starker CG Bogdanove AJ Voytas DF (2013) Transcription Activator-Like EffectorNucleases Enable Efficient Plant Genome Engineering Plant Physiology 161 20-27


Zhou H Liu B Weeks DP Spalding MH Yang B (2014) Large chromosomal deletions and heritable small genetic changes inducedby CRISPRCas9 in rice Nucleic Acids Research 42 10903-10914


Zuris JA Thompson DB Shu Y Guilinger JP Bessen JL Hu JH Maeder ML Joung JK Chen Z-Y Liu DR (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo Nat Biotech 33 73-80



Parsed Citations

Reviewer PDF

Parsed Citations


2

Targeted Mutagenesis Precise Gene Editing and Site-Specific Gene 14

Insertion in Maize Using Cas9 and Guide RNA 15

16

Sergei Svitashev1 Joshua K Young1 Christine Schwartz Huirong Gao S Carl Falco 17

and A Mark Cigan 18

19

Trait Enabling Technologies 20

DuPont Pioneer 7300 NW 62nd Avenue Johnston Iowa 50131 USA 21

22

23

24

25

Summary 26

27

Advances in double-strand break technologies modernize genome editing in maize28



3

Footnotes 29



32




36




40



4

41

ABSTRACT 42



















61



5

INTRODUCTION 62



























be achieved 89





6


























site sequence 117








7
















138

139



8

RESULTS 140

141
































9

















10


































11











226


in Maize 228





















12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


3

Footnotes 29



32




36




40



4

41

ABSTRACT 42



















61



5

INTRODUCTION 62



























be achieved 89





6


























site sequence 117








7
















138

139



8

RESULTS 140

141
































9

















10


































11











226


in Maize 228





















12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


4

41

ABSTRACT 42



















61



5

INTRODUCTION 62



























be achieved 89





6


























site sequence 117








7
















138

139



8

RESULTS 140

141
































9

















10


































11











226


in Maize 228





















12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


5

INTRODUCTION 62



























be achieved 89





6


























site sequence 117








7
















138

139



8

RESULTS 140

141
































9

















10


































11











226


in Maize 228





















12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


6


























site sequence 117








7
















138

139



8

RESULTS 140

141
































9

















10


































11











226


in Maize 228





















12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


7
















138

139



8

RESULTS 140

141
































9

















10


































11











226


in Maize 228





















12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


8

RESULTS 140

141
































9

















10


































11











226


in Maize 228





















12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


9

















10


































11











226


in Maize 228





















12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


10


































11











226


in Maize 228





















12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


11











226


in Maize 228





















12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


12





















these plants 267












13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


13



279






























14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


14















15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


15


































16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


16








3B) 357




361




17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


17


































18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


18





site in maize 398











19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


19



















425

426



20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


20

DISCUSSION 427










437














451









21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


21


































22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


22








496


























23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


23


































24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


24


experiments 552











563

CONCLUSIONS 564



















25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


25













593


595


















26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


26


































27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


27















precipitation 656



















28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


28














CA USA) 686

687

Plant Material 688










698








29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


29














717



















30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


30




























761






31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


31


























790

ACKNOWLEDGMENTS 791







32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


32



suggestions 798

799



33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


33

800








genome 812







experiment 819


821

822



34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


34

823



35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


35

824



36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


36


transformation 826


of Reads



Target Gene

LIG (Chr 2)


LIG-CR1 gRNA+Cas9 716854 33050 461

LIG-CR2 gRNA+Cas9 711047 16675 235

LIG-CR3 gRNA+Cas9 713183 27959 392

MS26 (Chr 1)


MS26-CR1 gRNA+Cas9 575671 10073 175

MS26-CR2 gRNA+Cas9 543856 16930 311

MS26-CR3 gRNA+Cas9 538141 13879 258

MS45 (Chr 9)

MS45-CR1 gRNA+Cas9 812644 3795 047

MS45-CR2 gRNA+Cas9 785183 14704 187

MS45-CR3 gRNA+Cas9 728023 9203 126


(Chr 5)

ALS-CR1 gRNA+Cas9 434452 9669 223

ALS-CR2 gRNA+Cas9 472351 6352 135

ALS-CR3 gRNA+Cas9 497786 8535 172





37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


37



829

830


of Reads


Reads


Target Gene

LIGCas-3

LIGCas-3 645107 12631 196

LIGCas-3 MS26Cas-2 579992 10348 178


MS26Cas-2

MS26 Cas 2 699154 17247 247

MS26Cas-2 LIGCas-3 717158 10256 143

MS26Cas-2 MS45Cas-2 613431 9931 162


MS45Cas-2

MS45Cas-2 503423 10034 199

MS45Cas-2 MS26Cas-2 480178 8008 167




38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


38

831


Target Sites

T0 Plant

qPCR Results


Integration


LIGCas-3

MS26Cas2


1 bp ins

1bp ins


2 nullnull 1bp ins

(T) 1bp del

1bp ins

(A)

1bp ins

(G) - - Yes Yes

MS26Cas-2


1bp ins


LIGCas-3

MS26Cas-2

MS45Cas-2


(T)

large

mut

1bp ins


large

mut Yes Yes


(T)

Wild-

type

1bp ins

(A)

1bp ins

(G)

1bp ins

(T)

large

mut No No




39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


39




841

Target Site





LIG




gRNA (RNA) 186

MDHCas9 event 1

gRNA (DNA) 025 -

gRNA (RNA) 012 -

MDHCas9 event 2

gRNA (DNA) 057 -

gRNA (RNA) 026 -

MDHCas9 event 3

gRNA (DNA) 046 -

gRNA (RNA) 035 -

MS26 MDHCas9

event 2

gRNA (DNA) 058 -

gRNA (RNA) 017 -




40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations


40


849


Events Analyzed


target site



Separate vectors


288 6 2 (07)


3 separate vectors


480 83 11 (25)




336 86 14 (41)



850

851


























































































































































Parsed Citations

Reviewer PDF

Parsed Citations

























































































































































Parsed Citations

Reviewer PDF

Parsed Citations

plant physiology preview. published on august 12, …...2015/08/12 · type ii system is the most...

Documents