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LARGE-SCALEBIOLOGYARTICLE

AMulti-purposeToolkittoEnableAdvancedGenomeEngineeringinPlants

TomášČermák1,ShaunJ.Curtin2,3,4,JavierGil-Humanes1,5,RadimČegan6,ThomasJ.Y.Kono3,EvaKonečná1,JosephJ.Belanto1,ColbyG.Starker1,JadeW.Mathre1,RebeccaL.Greenstein1,DanielF.Voytas1

1DepartmentofGenetics,CellBiology&DevelopmentandCenterforGenomeEngineering,UniversityofMinnesota,Minneapolis,MN554552DepartmentofPlantPathology,UniversityofMinnesota,StPaul,MN551083DepartmentofAgronomy&PlantGenetics,UniversityofMinnesota,St.Paul,MN551084Presentaddress:UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,CerealDiseaseLaboratory,StPaul,MN551085Presentaddress:CalyxtInc.,NewBrighton,MN551126DepartmentofPlantDevelopmentalGenetics,InstituteofBiophysicsoftheCAS,v.v.i.,CZ-61265,Brno,CzechRepublicCorrespondingauthor,DanielF.Voytas,voytas@umn.edu

Shorttitle:Toolkitforplantgenomeengineering

One-sentencesummary:Thisintegratedreagenttoolkitandstreamlinedprotocolsworkacrossdiverseplantspeciestoenablesophisticatedgenomeedits.

TheauthorresponsiblefordistributionofmaterialsintegraltothefindingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors(www.plantcell.org)is:DanielF.Voytas(voytas@umn.edu).

ABSTRACT

We report a comprehensive toolkit that enables targeted, specificmodificationofmonocot anddicotgenomes using a variety of genome engineering approaches. Our reagents, based on TranscriptionActivator-Like Effector Nucleases TALENs and the Clustered Regularly Interspaced Short PalindromicRepeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverseregulatory sequences to drive reagent expression. Vectors are optimized to create either single ormultiplegeneknockoutsandlargechromosomaldeletions.Moreover,integrationofgeminivirus-basedvectorsenablesprecisegeneeditingthroughhomologousrecombination.Regulationoftranscriptionisalso possible. Aweb-based tool streamlines vector selection and construction.One advantage of ourplatformistheuseoftheCsy-type(CRISPRsystemyersinia)ribonuclease4Csy4 andtRNAprocessingenzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstratetargeteddeletionsinuptosixgenesbyexpressingtwelvegRNAsfromasingletranscript.Csy4andtRNAexpression systems are almost twice as effective in inducing mutations as gRNAs expressed fromindividual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold byincorporatingtheTrex2exonuclease.Finally,wedemonstratethatCas9nickasesinducegenetargetingat frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. Thereagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotianatabacum),Medicagotruncatula,wheat(Triticumaestivum),andbarley(Hordeumvulgare).

Plant Cell Advance Publication. Published on May 18, 2017, doi:10.1105/tpc.16.00922

©2017 American Society of Plant Biologists. All Rights Reserved

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INTRODUCTION

Genome engineering is a rapidly emerging discipline that seeks to develop strategies and

methods for the efficient, targeted modification of DNA in living cells. Most genome

engineering approaches use sequence-specific nucleases (SSNs), such as TALENs and

CRISPR/Cas9reagents,tocreateatargetedDNAdouble-strandbreak(DSB)inagenome.DNA

repair then achieves the desired DNA sequence modification. DSBs are most frequently

repaired by non-homologous end joining (NHEJ), which can create insertion/deletion (indel)

mutations at the break site that knockout gene function. TALEN- or CRISPR/Cas9-mediated

geneknockoutshavebeenmadeindiverseplantspeciesamenabletotransformation(Brooks

etal.,2014;Fauseretal.,2014;Forneretal.,2015;Gaoetal.,2014;Christianetal.,2013;Liang

etal.,2014;Shanetal.,2013;Suganoetal.,2014;Xuetal.,2015;Zhangetal.,2014).Traitsof

commercialvaluehavealsobeencreatedbytargetedknockoutofselectedgenes(Wangetal.,

2014;Clasenetal.,2015;Haunetal.,2014;Lietal.,2015a,2012).Alternatively,DSBscanbe

repairedbyhomologydependentrepair (HDR),whichcopies information froma ‘donor’DNA

template. HDR typically occurs at lower frequencies thanNHEJ in somatic cells, and ismore

challengingtoachievebecause it requires introducing intotheplantcellboththedonorDNA

molecule and the SSNexpression cassette. As describedbelow, however, novel strategies to

deliver these reagents have recently realized some improvements in efficiency (Baltes et al.,

2014;Čermáketal.,2015;Fauseretal.,2012;Schimletal.,2014).

Since the development of TALENs and CRISPR/Cas9 reagents, improvements in the

technologyhaveoccurredatarapidpace.Forexample,indelfrequencieshavebeenincreased

by coupling SSNs with exonucleases (Certo et al., 2012; Kwon et al., 2012), altering gRNA

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architecture (Chenetal.,2013)orusingspecializedpromoters (Wangetal.,2015;Yanetal.,

2015).IncreasedspecificityhasbeenachievedusingpairedTALENorCRISPR/Cas9nickases(Ran

etal.,2013),truncatedgRNAs(Fuetal.,2014)ordimericCRISPR/Cas9nucleases(Guilingeret

al., 2014; Tsai et al., 2014). Other diverse applications have emerged, including targeted

regulation of gene expression, targeted epigenetic modification or site-specific base editing

through deamination (Russa and Qi, 2015; Kungulovski and Jeltsch, 2016; Zong et al., 2017;

Shimatanietal.,2017).

CRISPR/Cas9 is particularly useful for multiplexed gene editing, because target

specificityisdeterminedbyshortgRNAsratherthanproteinsthatmustbeengineeredforeach

newtarget(BaltesandVoytas,2015).Severalsystemsforsimultaneousexpressionofmultiple

gRNAshavebeendeveloped,andthesemostofteninvolvetheassemblyofmultiple,individual

gRNA expression cassettes, each transcribed from a separate RNA Polymerase III (Pol III)

promoter(Xingetal.,2014;Maetal.,2015;Lowderetal.,2015).MultiplegRNAscanalsobe

expressed from a single transcript. Such polycistronic mRNAs are processed post-

transcriptionally into individual gRNAs by RNA-cleaving enzymes. These enzymes include the

CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa (Tsai et al.,

2014), the tRNAprocessingenzymesnaturallypresent in thehost cells (Xie et al., 2015) and

ribozymes(GaoandZhao,2014).

One significant remaining challenge in plant genome engineering is achieving high

frequencygeneeditingbyHDR.Asmentionedabove,HDRrequiresintroducingintotheplant

cellbothadonorDNAmoleculeandaSSNexpressioncassette.DNAsequencemodifications

incorporated into the donor template are then copied into the broken chromosome. The in

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planta gene targeting (GT) approach stably integrates the donor template into the plant

genome.Thisensuresthateachcellhasatleastonecopyofthedonor,whichisreleasedfrom

thechromosomebyaSSN,makingitavailableforHDR(Schimletal.,2014;Fauseretal.,2012).

Anotherapproachinvolvesincreasingdonortemplateavailabilitybyreplicatingittohighcopy

numberusinggeminivirusreplicons(GVR)(Baltesetal.2014).GVRsmakeitpossibletocreate

precise modifications without the need to stably integrate editing reagents into the plant

genome(Cermaketal.2015).

Notallof themethodsandapproachesdescribedabovehavebeen implementedand

optimized for use in plants. Although there are several toolsets available for plant genome

engineering(Maetal.,2015;Xingetal.,2014;Lowderetal.,2015),theyarelimitedinthatthey

useasingletypeofDSB-inducingreagent(e.g.CRISPR/Cas9),asingletypeofexpressionsystem

(e.g.PolIIIpromotersforexpressionofgRNAs)ortheywerespecializedforasingleapplication

(e.g.geneknockouts).Here,weintroduceacomprehensive,modularsystemforplantgenome

engineering that makes it possible to accomplish gene knockouts, replacements, altered

transcriptionalregulationormultiplexedmodifications. Inaddition,theplatformcanbeeasily

upgraded and adjusted to accommodate novel reagents and approaches as they arise. Our

toolkitusesGoldenGatecloningforfastandeasyassemblyofgenomeengineeringconstructs

and flexibility in combining different functional modules for different applications. The

functional modules include TALE-based reagents (TALENs and TALE activators), CRISPR/Cas9

reagents (nucleases, nickases, catalytically inactive enzymes and activators), various gRNA

expression approaches (single and multi-gRNA expression systems using Pol III or Pol II

promoters), donor template plasmids for gene targeting, and a variety of other expression

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cassettes (Trex2, Csy4 and GFP). Further, a large set of promoters, terminators, codon

optimizedgenesandselectionmarkersfacilitateengineeringdicotormonocotgenomesusing

T-DNA or non-T-DNA vectors for Agrobacterium-mediated, biolistic or protoplast

transformation.Wealsoprovideauser-friendlywebportalwithprotocols,DNAsequencesof

all reagentsandweb-basedtoolstoassist inconstructdesign. Finally,wehavevalidatedour

reagentsinsixdifferentplantspeciesandreportthefirstuseofgenomeengineeringtocreate

modifiedMedicagotruncatulaplants.

RESULTS

Directandmodularassemblyofgenomeengineeringconstructs

Our plant genome engineering toolkit provides two sets of vectors, designated ‘direct’ and

‘modular’ (Figure1).Bothvectorsetscandeliver toplantcellseitherTALENsorCRISPR/Cas9

reagents for creating targeted DNA sequence modifications. The TALEN vectors are fully

compatiblewithourGoldenGateTALENandTALEffectorKit2.0(Cermaketal.,2011),andthey

use the highly active Δ152/+63 TALEN architecture (Miller et al., 2011). The CRISPR/Cas9

vectorsincludeeitherwheatorArabidopsisthalianacodon-optimizedversionsofCas9foruse

inmonocotsanddicots,respectively.BothversionsofCas9containasingleC-terminalnuclear

localizationsignal(NLS).

The direct cloning vectors (numbering 31) enable rapid construction of reagents for

making targeted gene knock-outs. They consist of vector backbones (T-DNA or non-T-DNA),

selectable marker expression cassettes and nucleases (i.e. Cas9 or the TALEN backbone).

However, they lack determinants for DNA targeting (i.e. gRNAs or TALE repeats). Single or

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multiplegRNAscanbeaddedinasinglestepusingaGoldenGateprotocol(Engleretal.,2008,

2009) (Figure 1A). Addition of TALENs uses a three-step Golden Gate protocol (see

SupplementalMethodsforassemblyprotocols).

ThemodularvectorsetusesGoldenGatecloningtocreatevectorsforadiverserangeof

geneeditingapplications(Figure1B,SupplementalMethods–Protocol5).Therearethreesets

ofmodularcloningvectors.ModulesetAcontainsready-to-useplasmids(currentlynumbering

61)withCas9orGFPexpression cassettes.ModuleAplasmidsarealsoavailable that canbe

used when assembling TALENs with our Golden Gate kit (Cermak et al., 2011). Module B

plasmids(currentlynumbering22)areusedeithertoaddsingleormultiplegRNAsinavariety

of expression formats or to add a second TALEN monomer. Module C plasmids (currently

numbering 22) may be used to add donor templates for gene targeting, additional gRNA

cassettesorother expression cassettes suchasGFP, Trex2orCsy4. Eachexpression cassette

hasa similararchitecture (SupplementalFigure1): a commonsetof restrictionenzymesites

are used that allow for easy swapping of regulatory elements (promoters, terminator

sequences)orcodingsequences.Ifareagentfromagivensetisnotdesired,thereareempty

modules to serve as placeholders and still allow for Golden Gate assembly. Finally, a

transformationbackbone canbe selected froma set (currently numbering 33) of T-DNAand

non-T-DNA vectors. Standard or geminivirus replicon (GVR) vectors are available with or

without one of the commonly used antibiotic selectable markers. For most applications,

creatingatransformationvectorusingthemodularvectorsetisatwo-stepcloningprocessthat

canbecompletedinfivedays;assemblyofTALEN-basedvectorsusuallyrequiresanadditional

cloningstep.

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Tofacilitateuseofourtoolkit,wedevelopedonlineresourcestoaidinvectorselection

and construct design (http://cfans-pmorrell.oit.umn.edu/CRISPR_Multiplex/). Our website

provides a complete list of both direct andmodular cloning vectorswith descriptions of key

features,DNAsequence filesand relevantprotocols fordownloading (seealsoSupplemental

DataSet1forthelistofvectors). Alsoavailableonlineisa‘vectorselection’toolthatusesa

dropdownmenutohelpidentifysuitableintermediaryplasmidsneeded(e.g.moduleA,BorC)

or to select a transformationbackbone.Thevector selection tool thenprovidesa link to the

DNA sequence file of the selectedplasmid that canbedownloaded inGenBank format. For

multiplexedgeneediting,our‘primerdesignandmapconstruction’tooltakesasinputaFASTA

filewiththegRNAtargetsequences.Alististhenpreparedofalltheprimersequencesneeded

tocreateavectorthatexpressesmultiplegRNAsusingourtRNA,Csy4orribozymeexpression

formats,andaGenBankfileiscreatedwiththeannotatedsequenceofthespecifiedgRNAarray

intheselectedplasmidbackbone.

TargetedmutagenesisofgenesinMedicagotruncatulausingdirectvectors

To test the functionality of the direct vectors, we designed a TALEN pair to target two

paralogous genes in Medicago (Medtr4g020620 and Medtr2g013650) (Figure 2A). The

Arabidopsis ortholog is known to play a role in phosphate regulation, and mutants hyper-

accumulatephosphate(Parketal.,2014).AT-DNAvectorwiththeTALENswasassembledusing

ourGoldenGateassemblyprotocol (SupplementalMethods–Protocol1B)and transformed

intoMedicago.ElevenT0plantswererecoveredandscreenedfortargetedmutationsatboth

Medtr4g020620andMedtr2g013650usingaPCRdigestionassay.TwoT0plants(WPT52-4and

WPT52-5)producedPCRampliconsthatwereresistanttorestrictionenzymedigestion(atboth

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targets inWPT52-4 and at one target inWPT52-5). PlantWPT52-4was self-fertilized, and T1

progenywerescreenedusingthesamePCRdigestionassay.Mutationswereconfirmedatboth

loci (Figure 2B).Mutations inMedtr4g020620were likely somatic, as only twoof the tested

plants produced digestion-resistant amplicons of varying intensity. However, mutations in

Medtr2g013650segregatedasexpectedforatraittransmittedthroughthegermline,andone

outofeighttestedplantswasahomozygousmutant.ThiswasconfirmedbyDNAsequencing,

whichrevealedfourdifferentMedtr4g020620deletionallelesinasingleplant,andanidentical

63bpdeletioninMedtr2g013650inthreeofthetestedT1plants(Figure2C).FromtheeightT1

plants,weidentifiedasingleplantwithmutationsinbothgenes.Thus,TALENsworkefficiently

inourdirectvectorsandinduceheritablemutations.

ExpressingmultiplegRNAsbyCsy4,tRNA-processingenzymesandribozymes

To harness one of the biggest advantages of the CRISPR/Cas9 system – the ability to target

multiple sites simultaneously – ourmodular cloning systemprovides vectors formultiplexed

genome editing. Typically in multiplexed editing, independent Pol III promoters are used to

drive expression of each gRNA. One limitation is that Pol III expression requires a specific

nucleotide in the first position of the transcript. Further, the size of the final array can be

cumbersome due to multiple, repeating promoter sequences. Processing polycistronic

transcriptscontainingmultiplegRNAsprovidesanalternativetotheuseofindependentPolIII

promoters.ThepolycistronicmessagecanbeproducedfromaPolIIpromoterthatisprocessed

eitherbytheCsy4protein,tRNAprocessingenzymesorribozymes(Nissimetal.,2014;Aebyet

al.,2010;Tangetal.,2016).PolIIpromotersprovideflexibilityintermsofspatialandtemporal

controlofexpressioninvivo,andtheyaremorelikelytoproducelongtranscripts,sincePolIIis

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not hampered by the presence of short internal termination sites, as is the case for Pol III

(Arimbasserietal.,2013).Wesoughttocomparetheefficiencyoftargetedmutagenesisusing

five different gRNA expression systems, including three different methods for processing

polycistronic gRNA transcripts (Figure 3A). All vectors were assembled using our modular

cloningsystemandusedtwogRNAstotargettwositesinthetomatoAUXINRESPONSEFACTOR

8AARF8Agene,apositiveregulatorofflowerdevelopmentandfruitset(Liuetal.,2014).

We created three vectors that use individual Pol III promoters to express each gRNA:

two AtU6 promoters, an AtU6 and At7SL promoter or AtU6 and At7SL promoters combined

with structurally optimized gRNA scaffolds (Chen et al., 2013). The remaining three vectors

produceasingle transcriptwithgRNAcassettesseparatedby20bpCsy4hairpins (Tsaietal.,

2014),77bptRNAGlygenes(Xieetal.,2015)or15bpribozymecleavagesites(inadditiontoa

synthetic ribozyme at the 3’ end of the transcript) (Haseloff and Gerlach, 1988; Tang et al.,

2016).ThankstotheadvantagesoverPolIIIpromotersmentionedabove,wechosetouseaPol

IIpromotertodrivetheexpressionofthepolycistronicgRNAs.SinceCas9wasexpressedfrom

a 35S promoter, we chose Cestrum Yellow Leaf Curling Virus promoter (CmYLCV) for gRNA

expressiontopreventuseofduplicatepromoters.TheCmYLCVpromoterdrivescomparableor

higher levelsofexpression than the35Sormaizeubiquitin (ZmUbi)promoters inbothdicots

andmonocots(Stavoloneetal.,2003),andtheintensityofGFPexpressedfromthispromoter

was similar to 35S-driven GFP in tomato protoplasts (Supplemental Figure 2). Final vectors

were co-transformed into tomato protoplasts along with a yellow fluorescent protein (YFP)

expressionplasmid. Transformationefficiencyapproximated60%, asdeterminedby counting

YFPpositivecells.

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Toassessmutationfrequencies, thetargetsitewasPCR-amplifiedfromDNAprepared

from the transformed protoplasts and subjected to Illumina DNA sequencing (Supplemental

Table1).AlloftheexpectedtypesofmutationsintheARF8Alocuswererevealedinallsamples

except for thenon-transformedcontrol (Figure3B,3C;SupplementalTable2; Supplemental

Figures 3-7). Interestingly, the overall frequency ofmutagenesiswas approximately two fold

higher with the Csy4 and tRNA expression systems compared to constructs with two Pol III

promoters.AlthoughtheoverallfrequencyofmutagenesisintheCsy4samplewasconsistently

higher than in the tRNA sample across three replicates, the difference was not statistically

significant(Figure3C,SupplementalTable3).Theribozymeshowedlowerfrequenciesofmost

mutation types, particularly at the 3’-most gRNA target site. We did not observe any

enhancement in overall mutation frequencies with the previously reported improved gRNA

architecture(Chenetal.,2013).Itisimportanttonotethatthemutationfrequencieswerenot

adjusted to account for the ~60% transformation efficiency, and therefore they are

underestimates.

DeletionbetweenthetwoCRISPR/Cas9cleavagesiteswasthemostcommonmutation

inallsamples(SupplementalFigure3).Shortinsertions/deletions(indels)ateitheroneorboth

cleavagesitesweremorethan10-foldlessabundant(SupplementalFigures3-6).Inversionsof

theinterveningsequenceanddeletionscombinedwithinsertionsweremorethan100-foldless

frequent(SupplementalFigure3).Mostinsertionsmappedeithertothetomatogenomeorto

thevector,coveringalmosteveryregionofthevectorsequence(SupplementalFigure7).

Cas9mostfrequentlycreatesablunt-endedDSBthreenucleotidesupstreamofthePAM

sequence(Jineketal.,2012).Inourexperiments,preciseligationofCas9-cleavedDNAwithout

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theinterveningsequencewouldresultinadeletionof85bp.Gainorlossofnucleotidesdueto

staggeredCas9cleavage(Jineketal.,2012;Lietal.,2015b)and/orexonucleaseactivitycould

result in shorteror longerdeletions.Strikingly,weobservedaveryhigh frequencyofprecise

ligation: on average, 83.5% of all reads with long deletions were deletions of 85 bp

(SupplementalFigure8).TheseresultssuggestthatifasinglegRNAwasused,mostinstances

ofNHEJrepairwouldnotresult inamutation.Thereforecreatingdeletionswithtwoormore

gRNAsshouldbeamoreefficientapproachtoachievetargetedmutagenesis.

Wefurthertestedtheeffectivenessofthepolycistronicexpressionsystemsusingeight

gRNAstargetingfourgenesfordeletionintomatoprotoplasts.Wealsomonitoredtheeffectof

thepositionofthegRNAinthearray.gRNAs#49-56(SupplementalTable4)wereassembled

directly intonon-T-DNAprotoplastexpressionvectors in twodifferentorders (Figure4A). In

onearray,thegRNAswereorderedfrom49to56,andtheorderwasreversed inthesecond

array. In addition, a vector expressing each gRNA from a separate Pol III promoter was

constructed. Each pair of gRNAs was designed to create a ~3 kb deletion to prevent

amplificationofthenon-deletedlociinsubsequentPCRanalysis(Figure4B).Thesevenvectors

were transformed into tomato protoplasts, and the frequencies of deletions induced by the

first,lastandoneoftheinternalgRNApairsineacharrayweremeasuredbyquantitativePCR

(Figure4C). Consistentwith thepreviousexperiment, theCsy4expression systemperformed

bestregardlessofthepositionofthegRNAinthearray.ThetRNAsystemwasthenextmost

effective,followedbytheuseofindividualPolIIIpromoters;theribozymesystemwastheleast

effective.gRNAsneartheendoftheCsy4andtRNAarrayswere lessefficient inmutagenesis

relative to gRNAs in earlier positions for themajority of tested loci, suggesting that position

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doeshaveaneffectongRNAactivity.Nevertheless, regardlessofpositioneffects,whenCsy4

was used, the frequencyof deletionswas as highor higher than theuseof individual Pol III

promoters,confirmingtheCsy4isthemostefficientsystemformultiplexedgeneediting.

Multiplexedmutagenesisintomato,wheat,andbarleyprotoplasts

WefurtherexploredtheeffectivenessoftheCsy4systemformultiplexingbytargetingsixgenes

in tomato (Solyc06g074350, Solyc02g085500, Solyc02g090730, Solyc11g071810,

Solyc06g074240andSolyc02g077390),threegenesinwheat(ubiquitin,5-Enolpyruvylshikimate-

3-phosphatesynthase-TaEPSPS,andMildewlocusO-TaMLO),andonegeneinbarley(Mildew

locusO-HvMLO).EachgenewastargetedfordeletionusingtwogRNAs.Toprovideexpression

in cereals, we created vectors with the maize ubiquitin (ZmUbi), switchgrass (Panicum

virgatum) ubiquitin 1 (PvUbi1) and switchgrass ubiquitin 2 (PvUbi2) promoters (Mann et al.,

2011).ThesequencesofPvUbi1andPvUbi2weremodifiedtoremoveAarI,SapI,andBsaIsites,

and themodifiedpromoterswere confirmed todrivehigh levelsofGFPexpression inwheat

scutella(SupplementalFigure9).Next,wedesignedCsy4arraysoftwelvegRNAstargetingthe

sixgenesintomato,sixgRNAstargetingthethreegenesinwheat,andtwogRNAstargetingthe

singlegeneinbarley.Intomato,the35SpromoterwasusedtoexpressCas9,andtheCmYLCV

promoter drove expression of the gRNAs. In wheat and barley, ZmUbi was used to drive

expression of Cas9, and the gRNA array was under control of PvUbi1. To test whether the

PvUbi1 promoter could be substituted with the significantly shorter viral CmYLCV for gRNA

expressioninmonocots,wealsodesignedavectorinwhichthetwogRNAstargetingthebarley

MLOgenewerecontrolledbytheCmYLCVpromoter.ThefinalvectorsexpressingCas9andthe

gRNA array were transformed into tomato, wheat and barley protoplasts, and PCR was

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conducted two days later to detect targeted deletions. Deletions of the expected size were

foundineachtargetedgeneandverifiedbyDNAsequencing(Figures5-7).BothCmYLCVand

PvUbi1-driven gRNA arrays induced targeted deletion of the barleyMLO gene with similar

efficiencies (Figure 7B). Thus, we confirmed that our system is efficient in simultaneously

creating multiplexed, targeted gene deletions in both dicots and monocots using up to 12

gRNAs.

Heritable,multiplexedmutagenesisinMedicagotruncatula

To demonstrate the efficiency of our Csy4multiplexing vectors inwhole plants,we used six

gRNAstotargetfourMedicagogenesthatencodenodule-specificcysteine-rich(NCR)peptides

(SupplementalFigure10).Morethan500genesmakeuptheNCRfamily,andtheirsmallsize,

sequence identity and overall number pose challenges to traditional knock-out/knock-down

platformssuchas transposableelements (Tnt1),RNA interferenceandchemicalmutagenesis.

Three gRNA pairs were designed to each delete one of three loci: NCR53, NCR54

(Medtr4g026818) andNCR55 (Medtr3g014705). TheNCR53 locus contains two homologous

NCR genes differing by several SNPs (Supplemental Data Set 2) and separated by

approximately 4 kb. The vector carrying the gRNA array, Cas9 expression cassette and bar

resistance gene was transformed into Medicago, and 46 phosphinothricin resistant T0

transformantswererecoveredandscreenedbyPCRanddirectsequencing.Forty-threeplants

hadmutations in inNCR53,eight inNCR54and12 inNCR55 (Table1,SupplementalTable5,

SupplementalDataSet2).Themutations includeddeletionsof theNCR53andNCR55genes,

resultingfromsimultaneouscleavageat twosites,aswellasshort indels inall three lociata

giventargetsite.Threeplantshadmutationsinallthreegenes,13weredoublemutantsand27

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hadmutationsinasinglegene.Despiteitsrepetitiveness,mutationsattheNCR53 locuswere

exclusively long deletions or inversions between the two cleavage sites in seven out of 46

plants.Nomutationswere recoveredat thegRNA3 site, likelydue to twoSNPs foundat the

targetsite.gRNA1,whichtargetsasiteatNCR53,hastwomismatchesintheseedregiontoa

siteatNCR54andaPAM-disruptingmutation.Similarly,gRNA2hastwomismatchestoasiteat

NCR54, one in eachof the 5’ and3’ endsof the seed region. Nooff-targetmutationswere

observedfromgRNA1andgRNA2at theNCR54 locus in the46plants, further indicatingthat

twomismatchesinthetargetsitearesufficienttopreventoff-targetactivity.

To confirm transmission of the mutations to the next generation, plant #16 (with

mutations inNCR53 andNCR54), plant#17 (withmutations inNCR53 andNCR55), plant#27

(with mutations in all three targets) and plant #40 (with mutations in NCR53) were self-

fertilized.SevenT1seedlingsfromeachT0parentwerescreenedbyPCRandDNAsequencing

formutations in the respective genes and for presence of thebar transgene (Supplemental

Figure 11A, B). Mutations identical to those observed in the T0 parents were found in the

NCR53 locusintheprogenyofallfourtestedplants(SupplementalFigure11A).Intwoofthe

fourplants(plant#17andplant#40),all testedprogenyonlyshowedthemutatedversionof

theNCR53 locus. Both thedoublemutant (plant #16) and the triplemutant (plant #27) also

transmittedtheexpectedmutationsinNCR54totheirprogeny,whereasonlythetriplemutant

(plant #27) and not the double mutant (plant #17) transmitted the mutations in NCR55.

Althoughwedidnotdetectthewild-typealleleofNCR53inT0plant#16orthewild-typeallele

ofNCR55intheT0plant#27,wild-typealleleswererecoveredintheT1progeny,suggestingthe

T0 plants might have been chimeric. Nevertheless, the T-DNA insertion segregated away in

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severalT1progenyofT0plants#16,#27(includingatriplemutant27-6)and#40(Supplemental

Figure 11B), confirming that the mutations in these plants were transmitted through the

germline rather than created de novo in the T1. Thus, we have been able to show that

mutationsineachofthegenesareheritable.Inaddition,weshowedthatmutationsinallthree

genescanbetransmittedsimultaneouslyfromatriplymutantparent(plant#27)toitsprogeny.

These results show the feasibility of using the Csy4 system to effectively recover heritable

mutationsinmultiplegenes.

Targeteddeletionof58kbinMedicagousingthetRNAandCsy4multiplexingsystems

To further evaluate the Csy4 and tRNA systems for creating chromosomal deletions in

Medicago,wedesignedanothersetofsixgRNAs(MPC1toMPC6)thattargeta58kbregionon

chromosome2(Figure8A).Theregioncontains fivecandidategenes identifiedbyagenome-

wideassociation study tobeassociatedwith symbioticnitrogen fixation (Curtinetal., 2017).

ThegRNAswereassembled(intheorderfrom1to6)intothedirectcloningT-DNAvectorsthat

useeitherCsy4ortRNAprocessingenzymes(seeSupplementalMethods–Protocols3Aand

3B),andaftertransformation,DNAfromrandomlyselectedcalliwastestedbyPCRforpotential

chromosomaldeletions. Primersweredesigned200 to300bpupstreamanddownstreamof

varioustargetsitesthroughoutthelocus(Figure8A,SupplementalTable6).Resultsfromthis

preliminaryassay revealedall fourpossibledeletions (detectableusing this setofprimers) in

the Csy4-transformed calli, whereas only the 27 kb deletion between gRNA sitesMPC1 and

MPC4wasdetected in thecalli transformedwiththetRNAmultiplexingreagents (Figure8B).

Amplicons generated from putative 58 kb deletions were sequence confirmed (Figure 8C).

While still in tissueculture,wepre-screened46T0plantlets fromeachof theCsy4and tRNA

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transformations for the 27 kb and 58 kb deletions. The putatively positive plantlets were

transferredtosoil,grownintomatureplantsandscreenedagainbyPCR.ThreeCsy4T0plants

wereidentifiedwiththe58kbdeletionandtwoplantswiththe27kbdeletion.ForthetRNAT0

plants,oneplanthadthe27kbdeletion(Figure8B).Thewild-typesequencewithinthe58kb

deletedregioncouldstillbeamplifiedwithadifferentsetofprimers,suggestingthatallplants

wereheterozygousorchimeric.Oneplant(WPT228-5)appearedtobeaT0homozygousmutant

for the 27 kb deletion, since we failed to recover a PCR amplicon from the wild-type allele

(Figure8B).

To test for heritable transmission of the deletions, T0 plants WPT228-28 (Csy4,

heterozygote or chimeric for the 58 kb deletion) and WPT227-28 (tRNA, heterozygote or

chimeric for the 27 kb deletion) were self-fertilized, and T1 plants were screened for the

segregating chromosomal deletions. Eight out of 12 T1 seedlings produced bands of the

expectedsizeforthe58kbdeletioninWPT228-28progeny,andsevenoutofsevenT1seedlings

showed bands expected for the 27 kb deletion inWPT227-28 progeny, confirming germline

transmissionintheselines(Figure8B).Inaddition,thewild-typeallelewasnotdetectedintwo

out of the eight WPT228-28 T1 seedlings, suggesting the two seedlings are homozygous

mutants. The bar transgene was not detected in three heterozygous WPT228-28 seedlings,

indicatingthe lossofT-DNAinsertionandconfirmingthatthemutationswerenotcreatedde

novo intheT1(Figure8B).Todeterminewhetherwecouldrecoverhomozygousmutants ina

transgene-freebackground,T1 plantWPT228-28-1–heterozygous for the58kbdeletionand

lackingtheT-DNAinsertion–wasself-fertilized,andsevenT2seedlingswerescreenedforthe

17

presence of the deletion. PCR and sequencing identified two homozygous and three

heterozygousnon-transgenicplants(Figure8B).

TheloweroverallmutationfrequenciescomparedwiththoseobservedintheNCRgeneediting

experimentare likelyduetothesignificantly largersizeofdeletionsbeingsought.An inverse

relationshipbetweenCas9-mediateddeletionfrequencyandsizehasbeenobservedpreviously

inbothmammalianandplantcells(Canveretal.,2014;Xieetal.,2015;Ordonetal.,2016).

EnhancingtargetedmutagenesiswithTrex2

To increase the frequency of NHEJ-induced mutations, sequence-specific nucleases can be

coupledwithexonucleasestopromoteendresectionandpreventprecise ligation.Previously,

co-expressionofthehuman3’repairexonuclease2(Trex2)withameganucleaseresultedina

25-fold enhancement in gene disruption frequencies in human cells (Certo et al., 2012). In

plants, overexpression of exonuclease 1 (Exo1) significantly enhanced the frequency of DSB

resectioninricecalli(Kwonetal.,2012),andTrex2increasedthemutagenesisfrequencywhen

co-deliveredtoplantcellswithameganuclease(Luoetal.,2015).Basedonthisdataandour

observation that the majority of Cas9-induced breaks are rejoined precisely, we tested the

effectonmutagenesisofexpressingTrex2withCas9andasinglegRNA.WeusedgRNAsthat

target two different sites in the tomato ANT1 gene and one site on the barleyMLO gene.

IndividualgRNAswereclonedunderthecontroloftheAtU6orTaU6promoters,combinedwith

a dicot or monocot optimized Trex2-P2A-Cas9 fusion, and tested in tomato and barley

protoplasts. We saw a modest 1.5-2.5-fold increase in indel frequencies in the presence of

Trex2comparedtothesampleswithoutTrex2inbarley(asmeasuredbytheT7EIassay)(Figure

18

9A).Similarresultswereobtainedintomato(asmeasuredbybothT7EIandDNAsequencing)

(Figure9A,B).Sequencingrevealeda trendtowards longerdeletions in theTrex2samples in

both tomato and barley (Figure 9B, C).Whereas no deletions >10 bp were detected in the

samples lacking Trex2, they represented 77% (tomato) and 100% (barley) of all mutations

recoveredinsamplesexpressingTrex2.Insertionsofupto42bpwerefoundinsampleswithout

Trex2 (Supplemental Figure 12) but not in the samples expressing Trex2, suggesting that

deletionswereinducedpreferentiallybythepresenceoftheexonuclease.

GenetargetingwithCas9nickases

Severalstrategieshavebeendevelopedtodecreasethefrequencyofoff-targetmutagenesisby

CRISPR/Cas9. Among these are the use of paired Cas9 nickases, which create a DSB by

introducingnicksonoppositeDNAstrands.Pairednickaseshaveon-targetcleavageefficiency

comparabletowildtypeCas9inhumancellsandplants(i.e.Arabidopsisandrice),andreduce

off-targetactivityupto1500-fold(Ranetal.,2013;Schimletal.,2014;Mikamietal.,2016).We

createdCas9nickasevariantsbymutatingthecatalyticresiduesintheconservedRuvC(D10A)

andHNH (H840A)nucleasedomains.ModuleA vectorswere then created thatexpressCas9

variantswithindividualorcombinedmutations(thelatterresultinginacatalyticallydeadCas9

enzyme).

Wewere interested inexploringwhetherpairedCas9nickasescanalsopromotegene

targeting(GT)inplantsusingapreviouslydescribedtobaccotransientGTassay.Inthisassay,a

defectivegus:nptII transgene is restoredby cleavage andhomologous recombination (Figure

10A) (Wright et al., 2005; Baltes et al., 2014). We designed a pair of gRNAs in PAM-out

orientationthatoverlappedby5bp(-5bpoffset)andshouldcreatea29bpoverhang(Figure

19

10A).TostimulateGT,thereagentsweredeliveredtotheplanttissueontheBeanyellowdwarf

virus (BeYDV) replicons (Baltesetal.,2014;Čermáketal.,2015)available inour toolkitasT-

DNAtransformationbackbones.Thereadoutwasthenumberofblue(GUSpositive)spotsand

intensityofstainingintobaccoleaves(SupplementalFigure13A).

WefirstcomparedtheGTfrequencies inducedbyasinglenickorDSB.Thenumberof

GTeventsinducedbysinglenickswasaround70%oftheGTeventsinducedbyaDSB(Figure

10B).NosignificantdifferencesinGTfrequencieswereobservedbetweentheD10AandH840A

nickases.Then,wecomparedtheGTefficienciesusingpairednickasesthatinducedoublenicks

resulting in 29 bp 5’ overhangs (AtD10A) and 3’ overhangs (AtH840A). We observed 2-fold

increaseinGTwith5’overhangscomparedto3’overhangs.ThenumberofGTeventsinduced

bydoublenickscreating5’overhangswasevenhigherthanthenumberofGTeventsinduced

bythewild-typeCas9nuclease.Thisis inagreementwithobservationsinhumancells(Ranet

al.,2013).

ToconfirmthenatureoftheGTeventsonthemolecularlevel,weextractedDNAfrom

infiltrated tobacco leaves and PCR amplified both junctions of the inserted sequence

(Supplemental Figure 13B). All samples except the one that was infiltrated with the vector

expressingnon-cuttinggRNAs,yieldedproductsofexpectedsizeforbothjunctions.Sixtoten

bacterial clones for each junction from each sample were analyzed by sequencing, which

revealed precise integration of the donor template in all clones (Figure 10C). These data

suggestthattheoverallfrequencyandprecisionofGTinducedbynickasesiscomparabletothe

nuclease-basedapproach,whichwehavepreviouslyshowncanbeusedtoefficientlyrecover

plantswithheritablemodificationscreatedbyGT(Baltesetal.,2014;Čermáketal.,2015).

20

To see whether the nickase approach is applicable for GT in a monocot species, we

targetedanin-frameinsertionofaT2A-GFPcassetteintothethirdexonoftheubiquitingenein

wheat scutella, usingWheat dwarf virus (WDV) replicons as described in Gil-Humanes et al.

(2017). We observed GFP expression consistent with insertion into the ubiquitin locus

(Supplemental Figure 14A-C), suggesting the feasibility of this approach across different

species.

DISCUSSION

Genome engineering promises to advance both basic and applied plant research, and we

developed a comprehensive reagent toolkit to make the latest genome engineering

technologiesavailable to theplant sciencecommunity.BothTALENsandCRISPR/Cas9canbe

easilycombinedwithavarietyofregulatorysequences,transformationvectorsandotherDNA

modifyingenzymes,making itpossibletorapidlyassemblereagentsnecessarytomakesingle

andmulti-geneknockoutsaswellasgenereplacements.

Usingour flexible,modularsystemweevaluated fourdifferentmulti-gRNAexpression

strategiesintomatoprotoplaststoachievemultiplexedediting.ApolycistronicgRNAtranscript,

when processed by the bacterial Csy4 ribonuclease, resulted in almost two times higher

mutation frequencies compared with the commonly used strategy in which each gRNA is

expressedfroman individualPol IIIpromoter.Further, thepositionofthegRNAs inthearray

hasonlyamodesteffectonactivity,andthelastgRNAsinanarrayofeightperformcomparably

togRNAsexpressedindependentlyfromPollIIIpromoters.Althoughthefrequenciesobserved

with Csy4 were comparable to the previously described tRNA processing system (Xie et al.,

21

2015),wefavortheCsy4systembecauseitconsistentlygaveushigherfrequenciesofdeletions

intomatoprotoplasts,andthemulti-gRNAtranscriptsareshorterandlessrepetitive(i.e.Csy4

recognitionsitesare20bpvs.77bpfortRNAs).AlthoughtheCsy4systemrequiresexpression

oftheCsy4ribonucleaseintheplantcell,andnoextrafactorsarerequiredfortRNAprocessing,

theCsy4geneisrelativelyshort(561bp=187aa),andwetypicallyexpressitasP2Afusionto

Cas9withoutanyobservablephenotypicconsequencestotransgenicplants.

Although we have previously shown that gRNA transcripts processed by ribozymes

efficiently induce mutagenesis in rice, tobacco, and Arabidopsis (Tang et al., 2016), of the

polycistronicRNAprocessingmechanismstestedinthepresentstudy,ribozymesresultedinthe

lowestmutation frequencies. One difference between the two studies is that we expressed

Cas9 and thegRNAarray fromseparatepromoters,whereasTangetal. (2016)useda single

transcription unit to express both the Cas9 and the gRNAs. Furthermore, the frequency of

deletionscreatedbytwogRNAswasnotdirectlymeasuredinthepreviousstudy.Itispossible

that coordinate expression ofCas9 and gRNAs from a single transcription unit, which is not

possible in the current version of our cloning systemdue to itsmodular structure,might be

essentialtoachievehighergeneeditingfrequenciesusingribozymeprocessing.Whetherthisis

true remains to be elucidated. Nevertheless, we demonstrate Csy4 and tRNA expression

systemsefficientlyprocessmulti-gRNAtranscriptsandenablehighefficiencymultiplexedgene

editing.

AnalysisofmutationsresultingfromcleavageofasinglegenewithtwogRNAsrevealed

that approximately 85% of DSBs were precisely ligated. This is consistent with the high

frequencyofpreciserepairobservedinmouseandhumancells(Lietal.,2015b;Geisingeretal.,

22

2016).SinceDSBsinducedbyasinglegRNAarealsolikelyaccuratelyrepaired,wefavortheuse

of two gRNAs to induce loss-of-function mutations. Precise repair can be used to your

advantagetomakethemutationofchoice,andtherearenoconcernsaboutcreatingfunctional

geneisoformsthroughnon-frameshiftindelsoralternativesplicing.Furthermore,thetargeted

deletionscanbedetectedbyPCR,makingthescreeningformutantssignificantlyeasier.Thisis

particularly valuable, if not essential, when simultaneously mutagenizing several genes, as

screening for loss of restriction enzyme sites, T7 assays or direct DNA sequencing is time

consuming and costly. Targeted deletions might also be valuable for editing non-coding

sequences, including promoters and regulatory sequences,where short indelmutationsmay

not be effective. Moreover, engineering complex traits will likely require an efficient

multiplexededitingplatform.Hereweshowthatcombinationsofdifferentgenedeletionscan

be induced in a single experiment using a single vector, highlighting the potential of this

approachtospeedupcandidategeneevaluation.UsingourCsy4-basedsystem,wesuccessfully

deleteduptosixgenes intomato,wheat,andbarleyprotoplastsand inMedicagotruncatula

plants,demonstratingtheversatilityofthissystemacrossavarietyofplantspecies.Finally,the

high level of precision inNHEJ repair also opens up an opportunity for targeted and precise

geneinsertion/replacement,independentofHDR(Geisingeretal.,2016).

As an alternative to knocking out geneswith twoormore gRNAs,we also show that

Trex2, in combination with Cas9, increases the length and frequency (2.5 fold) of NHEJ-

mediateddeletions inbothtomatoandbarley.AsinglestudythatcombinedTrex2withCas9

forgeneeditinginhumancellsreportedasimilar2.5-foldincreaseinmutagenesis(Charietal.,

2015). When Trex2 was used in combination with TALENs, a 3-4-fold enhancement was

23

observed(Certoetal.,2012;Luoetal.,2015),whereasa25-foldincreasewasachievedwhena

meganucleasewasusedastheDSB-inducingagent(Certoetal.,2012).Thedifferencesinfold-

enhancementbetweenthethreetypesofnucleasesmaybecausedbydifferentlevelsofTrex2

activity on different types of DNA ends induced by Cas9 (blunt), TALENs (5’ overhang) or

meganucleases(3’overhang,thepreferredsubstrateofTrex2).Interestingly,Certoetal.(2012)

detectedabias towards smalldeletionswhen they combinedTrex2withmeganucleasesand

TALENs,butthestudyofCharietal.(2015)aswellasourdataindicatethatthedeletionlength

increaseswhenTrex2isusedwiththeCRISPR/Cas9system.Nevertheless,deletionsseemtobe

induced preferentially in the presence of Trex2, regardless of the type of nuclease used to

createtheDSBsincethelowerfrequencyof insertionscomparedwiththenoTrex2control is

consistentinallstudies.

Cas9nickasesarefrequentlyusedbecausetheyreducetheriskofoff-targetmutations

(Ranetal.,2013;Choetal.,2014;Shenetal.,2014;Mikamietal.,2016).Singlenickasesdonot

inducedetectablelevelsofon-targetNHEJ-mediatedmutagenesis(Congetal.,2013;Ranetal.,

2013; Fauser et al., 2014), whereas paired nickases, which create two adjacent nicks in the

targetsequence,inducemutationsatefficienciescomparabletonativeCas9(Ranetal.,2013;

Schimletal.,2014;Mikamietal.,2016).Pairednickasesalsoefficientlyinducegenetargetingin

humancells(Malietal.,2013;Ranetal.,2013),andherewedemonstratethatpairednickases

constructed using our modular system induce gene targeting in tobacco and wheat with

efficienciescomparabletothenuclease.Consistentwiththedatafromhumancells(Malietal.,

2013;Ranetal.,2013),pairednickasesthatcreate5’overhangs inducedGTmorefrequently

thanthosecreating3’overhangs.Inaseparatestudy(Fauseretal.,2014),singlenickaseswere

24

foundcapableofpromotingGTinArabidopsisatfrequenciesexceedingthenuclease;however

weobservedthatGTfrequencieswithsinglenickaseswereabovebackgroundinbothtobacco

andwheat,buttheywerelowerthantheGTfrequencyachievedwiththenucleaseandpaired

nickases.ThehighfrequencyofGTobservedbyFauseretal. (2014)withasinglenickmay in

partbeduetheproximityofthedonortemplate,whichwasintegratedintothechromosome

adjacent to thenick site. Indeed, significantly lowerGT frequencieswereobserved inhuman

cells when single nickases were used with extrachromosomal donors (Ran et al., 2013),

although other factors such as donor architecture and cell type could contribute to the

observeddifferencesbetweenstudies.Nevertheless,weshowthatpairednickases,whichhave

beenshowntohaveminimaloff-targeteffects inplants(Mikamietal.2016),canbeusedfor

efficientGTinbothmonocotsanddicots.

Anumberofdifferenttoolkitsforplantgenomeengineeringhavebeenpublishedwithin

thepasttwoyears(Xingetal.,2014;Xieetal.,2015;Maetal.,2015;Lowderetal.,2015;Zhang

etal.,2015;Liangetal.,2016;Vazquez-Vilaretal.,2016;Kimetal.2016;Ordonetal.,2016);

however,allhavelimitations:mostdonotofferflexibilityinthegeneeditingplatform(TALENs

or CRISPR/Cas9); some reagents are optimized for a non target organism (e.g. humans);

typicallythereislittlechoiceinpromotersforexpressingtargetingreagents;mostsystemsare

limited to a single vector type (e.g. T-DNA); applications are limited to NHEJ-mediated gene

knockouts;mostreagentsarevalidatedinonlyoneortwomodelspecies.Althoughtwostudies

havemadetheirapproachescompatiblewithawidervarietyofvectorsandreagents,suchas

theD10ACas9nickaseandCas9-basedtranscriptionalactivatorsandrepressors(Lowderetal.,

2015;Vazquez-Vilaretal.,2016),theyremainlimitedinotheraspects.Forexample,theLowder

25

et al. 2015 toolkit limits gRNA expression to Pol III promoters, requires an 8-day assembly

process,andlackscross-compatibleandadaptableexpressionvectors;theVazquiez-Vilaretal.

2016 toolkit requires a time-consuming binary cloning approach and lacks substantial

evaluation of the multiplexed gene editing vectors. In comparison, our plant genome

engineeringplatformprovides theuserwithanoption to choose thedesired reagent froma

comprehensivesetofexpressioncassettesincludingTALENs,TALE-VP64,TALE-VPR,Cas9,D10A

Cas9 nickase, H840A Cas9 nickase, dCas9, dCas9-VP64 and dCas9-VPR, GFP, Trex2 and Csy4

withversionscodonoptimizedforuseindicotsormonocots.ForCas9-basedgenomeediting,

the toolkit enables assembly of vectorswith all gRNA expression systems described to date,

including two dicot and four monocot Pol III promoters. Furthermore, multiplexed systems

basedonCsy4,tRNAandribozymesareavailablethatcanbecombinedwithPolIIpromoters.

All reagents can be assembled into T-DNAor non-T-DNA vectorswith orwithout one of the

threemost common plant selectablemarkers driven by dicot ormonocot promoters. Faster

direct or flexiblemodular cloning protocols are available.While previous studies focused on

evaluating reagents in the model species Arabidopsis, rice and tobacco, here we provide

evidence that inaddition to tobacco,our system is functionalandeffective in two important

dicotandtwomonocotcropspecies.

Tothebestofourknowledge,ourtoolkitisthefirstdesignedtofacilitategenetargeting

through homologous recombination. We include GVR-based vectors, which were first

demonstrated to work in proof of concept experiments in tobacco (Baltes et al., 2014) and

morerecentlytoeffectivelyintroduceheritabletargetedinsertionsintothetomatogenomeat

frequencies3-9-foldhigherthanstandardT-DNA(Čermáketal.,2015).WehavealsousedWDV

26

repliconstotargetinsertionofaselectablemarkerintothewheatubiquitinlocus(Gil-Humanes

etal.,2017),andhereweshowGVRsworkincombinationwithpairednickases.Moreover,our

modularsystemisalsocompatiblewiththeinplantagenetargetingapproachdescribedearlier

(Fauser et al., 2012). An integral element in a gene targeting strategy is the DNA donor

template,whichcarriescustommodificationsbeingintroducedinthetargetgenome.Allofthe

module C plasmids in our toolkit include a unique restriction site for insertion of the donor

template,andweprovideprotocolsforassemblyofthedonortemplateplasmidsforboththe

repliconand inplantagenetargetingmethods (seeSupplementalMethods–Protocol4).All

together, our toolkit enables the most efficient plant gene targeting methods currently

available.

In addition to the commonly used 35S and maize ubiquitin promoters, the current

versionofourcloningsystemincludessevenotherdicotandthreemonocotpromoters.These

includeconstitutiveviral,bacterial(CmYLCV,M24,FMV,nos)orplant(AtUbi10,OsAct1,PvUbi1,

PvUbi2)promoters,whichprovidearangeofexpressionstrengthsinavarietyofplantspecies

(Stavoloneetal.,2003;Sahooetal.,2014;Maitietal.,1997;Norrisetal.,1993;McElroyetal.,

1990; Mann et al., 2012). We have also included the Arabidopsis thaliana Ec1.2 and YAO

promoters, which are egg cell and embryo sac/pollen specific, respectively. Although

Arabidopsiswasnotourtargetspeciesinthisstudy,thesetwopromotershavebeenshownto

enhancemutagenesis inArabidopsiswhenused todriveCas9expression (Wanget al., 2015;

Yan et al., 2015). In our toolkit, these promoters can drive either Cas9 expression or the

polycistronic gRNA arrays. Due to the universal structure of the modular vectors, new

combinations of promoters and coding sequences canbe easily added. The versatility of the

27

toolkit is further increased by its optimization for Golden Gate assembly, one of the most

popular DNA assembly methods. We have made the T-DNA backbones derived from the

commonly used pCAMBIA vectors free of most relevant type IIs restriction sites, AarI, BsaI,

Esp3I and SapI. AarI, SapI and BsaI sites were also removed from the CmYLCV, PvUbi1 and

PvUbi2promoters,andtheactivityofthemodifiedpromoterswasverifiedinplantstoensure

these modifications do not affect expression levels. Since the Golden Gate modules can be

easily modified to contain other genes, the system can be adopted for applications beyond

genomeengineering,suchassyntheticbiology.

Finally, theability toalter transcription levelsofendogenousgenes isoftendesirable.

TranscriptionalactivationdomainshavebeenfusedtobothTALEsandCas9toenabletargeted

gene activation (Lowder et al., 2015; Vazquez-Vilar et al., 2016). Although these custom

transcriptionfactorshaveproveneffective (Liuetal.,2013;Piateketal.,2015;Lowderetal.,

2015; Vazquez-Vilar et al., 2016), they havemostly been testedon reporter genes drivenby

syntheticorminimalviralpromoters.Therefore,theincreaseinexpressionlevelsmightnotbe

sufficient to inducephenotypicchangeswhenendogenousgenesare targeted (Lowderetal.,

2015).Toovercomethemodestlevelsofgeneactivation,Chavezetal.(2015)recentlyshowed

thatfusionsofdCas9orTALEproteinstoastrongtranscriptionalactivatorVPRmediateupto

320-fold higher activation of endogenous targets compared with fusions to VP64 alone.

Althoughfurtheroptimizationofartificialplanttranscriptionalregulatorsisneeded,atpresent,

weprovidemoduleswithfusionsofbothVP64andVPRactivatordomainstoTALEsanddCas9

fortranscriptionalactivation,aswellasmoduleswithdCas9only,whichhasbeenshowntobe

28

equallyeffectiveintranscriptionalrepressionasdCas9fusionstorepressordomains(Piateket

al.,2015;Vazquez-Vilaretal.,2016).

Inconclusion,wewouldliketonotethatduetoourtoolkit’smodulardesign,itcanbe

easily updatedwith new functions, andwe intend to incorporate new tools as they become

available.Giventheeaseandspeedofreagentconstructionandtheavailabilityofuser-friendly

protocols andonline tools,wehopeourplatformwill accelerateprogress inplant functional

genomicsandcropimprovement.

Methods

Directandmodularvectorlibraryconstruction

Direct TALEN cloning vectors pDIRECT_37-39J were first constructed by Gibson assembly

(Gibsonetal.,2009)ofPCRproductscontainingpartsofpCLEAN-G126backbone(Tholeetal.,

2007,GenBankacc.EU186082), selectionmarker cassettes, theOsADH 5’UTRamplified from

riceOryza sativa cv.Nipponbare genome (Sugio et al., 2008), 35S promoter,δ152/+63 TALE

scaffoldsincluding3xFLAGepitopeandSV40NLS,Esp3IflankedccdBcassette,BsaIflankedLacZ

cassette, ELD andKKRheterodimeric FokI variants (Doyonet al., 2011) andArabidopsis heat

shock protein 18.2 (HSP) terminator amplified from the A. thaliana genome (Nagaya et al.,

2010). In theprocess,Esp3I recognitionsiteswere removed fromthenptII geneand theELD

FokICDS,BsaIfromtheccdBgeneandtheBglIIsitefromtheHSPterminator.Uniquerestriction

siteswereinsertedtoflankthepromoterandterminatorinthedualTALENexpressioncassette.

TALE sequences frompTALplasmids (Cermaketal., 2011)wereusedand19bpof sequence

immediatelyupstreamofthefirstEsp3IsiteinTALEN1anddownstreamofthesecondBsaIsite

29

inTALEN2werere-codedtoincludeuniqueprimerbindingsitesforcolonyPCRandsequencing

of both TALE repeat arrays. pCAMBIA-based vectors pDIRECT_21-23J were constructed

accordingly,excepttheywerefirstmadewithanAscIandSacIflankedtheccdBcassette,later

replaced with the AscI/SacI fragment from the pCLEAN-based vectors containing the dual

TALEN expression cassette; and unique restriction sites were inserted to flank the selection

marker gene. For expression in monocots, the ZmUbi1 promoter was PCR amplified from

pAHC25(ChristensenandQuail,1996)andclonedbetweenAscIandSbfIsitesofpDIRECT_21J

andpDIRECT_23Jtoreplacethe35SpromoterandcreatepDIRECT_25KandpDIRECT_26K.To

createthefirstmoduleplasmids,theBsaILacZcassetteinpTC102(aderivativeofpDIRECT_37J

with translation initiation sequence optimized for dicots) was replaced with a second Esp3I

ccdBcassttetocreatepTC163.ThefirstandsecondTALENcassetteswerePCRamplifiedfrom

pTC163(splittingtheP2AsequencebetweenthetwocassettesandremovingtheAarIsitefrom

it)andaBaeIsitecontainingfragmentwasamplifiedfrompTC130(Čermáketal.,2015),using

threepairsofprimerscontainingAarIsiteswithdistinct4bpoverhangs.Thefirstthreemodule

plasmidspMOD_A1001,pMOD_B2000andpMOD_C0000were constructedby insertingeach

ofthesethreePCRfragmentsintoBsaXIandPmlI-linearizedpTAL3(Cermaketal.,2011)using

Gibsonassembly.TogeneratethefirstCRISPR/Cas9modulespMOD_A0101andpMOD_B2515,

PCRfragmentscontaining35Spromoter,AtCas9genecodon-optimizedforA.thaliana(Fauser

et al., 2014) and the HSP terminator; or the AtU6 gRNA cassette were inserted into AarI

linearized pMOD_A1001 and pMOD_B2000 by Gibson assembly. Similarly, an At7SL gRNA

cassette was inserted into NaeI and XhoI cut pMOD_C0000 to create pMOD_C2516. All

remaining module plasmids were derived from these initial plasmids through replacing the

30

genecassettesandpromotersusingtheuniquerestrictionsitesAscI,SbfI,XhoIandSacI,orin

case ofmulti-gRNAplasmids, throughGoldenGate assembly of PCR products containing the

specific elements. TaCas9 was codon optimized for wheat and Gibson assembled from 9

gBlocks (Integrated DNA Technologies, Coralville, USA). Cas9 nickases and catalytically dead

variantsweremadebyGibsonassemblyofPCRproductscontainingthespecificmutationsinto

the Cas9 nuclease modules. Cas9 and TALE transcriptional activators were constructed by

Gibsonassemblyof fragments intoTALENanddCas9containingmodules.TheVPRactivators

werebuiltfirst,usingthetripartiteVPRtranscriptionactivationdomaindescribedinChavezet

al.(2015).TheVP64activatorswerederivedfromtheVPRmodulesbydeletingthep65andRTA

domains by restriction cloning. The Csy4 genes, codon-optimized for dicots (tomato + A.

thaliana) and monocots (wheat and rice), were synthesized as gBlocks along with the P2A

ribosomal skippingsequenceandGibsonassembled intoSbfIandSalIdigestedpMOD_A0101

and SbfI andBstBI digestedpMOD_A1510, respectively.Trex2was cloned similarly, except it

wasPCRamplifiedfrompExodusCMV.Trex2plasmid(Certoetal.,2012)containingthemouse

versionofthegenewhichwepreviouslyobservedhadrobustactivityinplantcells.TheCmYLCV

(withouttheAarIsite)andFMVpromotersweresynthesizedasgBlocks.ThePvUbi1andPvUbi2

promoterswereamplifiedfrompANIC10A(Mannetal.,2012)eachasasetofoverlappingPCR

fragments to enable removal of AarI, BsaI and SapI sites throughGibson assembly. TheNos

promoter, rbcsE9 and 35S terminators were amplified from pFZ19 (Zhang et al., 2010) and

pCAMBIAvectors.TheremainingpromoterfragmentswereobtainedbyPCRamplificationfrom

therespectivegenomicDNAs.ThetRNAGlysequencesusedtocreatethemultigRNAconstructs

were amplified from tomato cv. MicroTom genomic DNA, while the Csy4 and ribozyme

31

sequencesweresynthesizedaspartsofprimersforGoldenGateassembly.Finally,theempty

modulespMOD_A0000andpMOD_B0000werecreatedby ligatingannealedoligonucleotides

carryinguniquerestrictionsitesintotheAscIandSacIsitesinpMOD_A0101andAscIandAgeI

sitesinpMOD_B2103.TheT-DNAtransformationbackboneswerederivedfrompCAMBIA1300,

2300and3300vectorsbyGibsonassemblyoftheCmR/ccdBselectioncassetteamplifiedfrom

pMDC32 (Curtis and Grossniklaus, 2003) using primers carrying AarI sites specifying 4 bp

overhangsdesignedtoacceptinsertsfromthethreemodules,intoHindIIIandBamHIdigested

pCAMBIA backbones. In addition, pTRANS_210d-250d aremodified versions of the resulting

vectors with AarI, BsaI, Esp3I and SapI sites removed from the vector backbone by Gibson

assembly of backbone fragments overlapping at the mutated sites. pTRANS_250d and

pTRANS_260dcontainPvUbi1 andPvUbi2promoters thatweredirectlyassembled into these

vectors as described above. All non-T-DNA transformation backbones are derived from

pTRANS_100.ThisvectorwascreateddenovobyGibsonassemblyof twoPCRproducts,one

containingthespectinomycinresistancegeneandbacterialoriginofreplicationamplifiedfrom

pTC14 (Cermak et al., 2011) and the other containing the AarI flanked CmR/ccdB selection

cassette from the T-DNA transformation vector pTRANS_210. The selectionmarker cassettes

were then inserted into this vector by restriction cloning from the T-DNA transformation

backbones. The GVR transformation vectors were built by Gibson assembly from modified

pCAMBIAbackbone(withoutBsaIandEsp3Isites)andgeminiviruselementsdescribedinBaltes

etal.(2014),Čermáketal.(2015)andGil-Humanesetal.(2017)AllCRISPR/Cas9directcloning

andCas9onlyvectorswereassembledbyGoldenGateassemblyfrommodulesA,BandC.To

32

createtheCas9onlyvectors,thetransformationbackbonesweremodifiedtoaccepttheinsert

fromasinglemodule.

GeneeditinginMedicagotruncatula

FortargetedmutagenesisoftheMedtr4g020620andMedtr2g013650Agenes,apairofTALENs

wasassembledintopDIRECT_39JusingProtocol1B(SupplementalMethods)resultinginpSC1

and transformed into an Agrobacterium tumefaciens strain EHA105 for whole-plant

transformation of theMedicago truncatula accession HM340 using an established protocol

(Cossonet al., 2006). The resultingplantswere screened for putativemutationsusing a PCR

digestionassaywithprimerslistedinSupplementalTable6andtheHaeIIIrestrictionenzyme.

Thedigestion-resistantproductswereclonedandseveralclonesweresequencedtoidentifythe

mutations. For combinatorial deletion of NCR genes, the six gRNAs were assembled into

pDIRECT_23C along with the CmYLCV promoter using Protocol 3A (SupplementalMethods)

resulting in pSC2 and transformed into Agrobacterium tumefaciens EHA105. To detect the

targetedgenedeletionsandshort indelsateachgRNAsite,thethreelociwerePCRamplified

fromT0andT1plantsusingprimerslistedinSupplementalTable6.ThePCRproductswerefirst

directly sequenced. PCR products from samples containing both the long deletion and non-

deletionalleleswere gel purifiedbefore sequencing and sequenced separately. Samples that

showedoverlappingsequencetracesstartingateithergRNAsiteindicativeof indelmutations

wereclonedandseveralclonesweresequencedtoidentifythetypeofmutations.Fortargeted

deletionofthe58kbregiononchromosome2,thesixgRNAswereassembledasCsy4ortRNA

arrays driven by the CmYLCV promoter into pDIRECT_23C using Protocol 3A and 3B

(SupplementalMethods)yieldingpSC3andpSC4,respectively,andtransformedintoMedicago

33

truncatulaaccessionHM340asabove.Targeteddeletions intheresultingT0,T1andT2plants

weredetectedusingprimerslistedinSupplementalTable6,clonedandsequenced.

Determiningtheefficiencyofmulti-gRNAexpressionsystemsintomatoprotoplastsbydeep

sequencing

To build vector pTC303, expressing the two gRNAs from AtU6 and At7SL promoters; and

pTC363expressingeachgRNAfromanindividualAtU6promoter,thegRNAspacerswerefirst

clonedintopMOD_B2515(moduleBwithAtU6promoter,usedforbothpTC303andpTC363),

pMOD_C2515(moduleCwithAtU6promoter,usedforpTC363)andpMOD_C2516(moduleC

with At7SL promoter, used for pTC303) using Protocol 2A (Supplemental Methods). The

resulting plasmids were assembled together with pMOD_A0101 into the pTRANS_100

protoplast vector using Protocol 5 (SupplementalMethods). pAH750, expressing the gRNAs

with structurally optimized scaffolds from AtU6 and At7SL promoters was constructed

accordingly,exceptthegRNAspacerswereclonedintopMOD_B2515bandpMOD_C2516b,the

respectivemoduleBandCplasmidsthatcarrytheoptimizedgRNArepeats.TheCsy4,tRNAand

ribozymeexpressioncassetteswerecreateddirectlyinprotoplastvectorspDIRECT_10C(Csy4)

andpDIRECT_10E(tRNAandribozyme)usingProtocol3A,3Band3C(SupplementalMethods),

resulting in pTC364 (Csy4), pTC365 (tRNA) and pTC366 (ribozyme). Next, protoplasts were

isolated from tomato cv. MicroTom as described in Zhang et al. (2012), with minor

modifications.Leavesfrom1-2sterilegrown(26°Cwith16hlight)~4weeksoldplantletswere

slicedintothinstripswithasharprazor,transferredtotheenzymesolution(1.0%cellulaseR10,

34

0.25%macerozymeR10, 0.45Mmannitol, 20mMMES, 1xMS salts and0.1%bovine serum

albumin)andincubated15hat25°Cand40rpminthedark.Thedigestedproductwasfiltered

througha40-µmcellstrainer(Falcon)andpipetteddirectlyontopof0.55Msucrosesolution.

TherestoftheprotocolfollowedZhangetal.(2012),exceptW5solution(2mMMES,154mM

NaCl,125mMCaCl2and5mMKCl)wasusedaswashingbuffer.TheCRISPR/Cas9constructs

were co-transformed into ~200,000 cells along with the YFP expression plasmid pZHY162

(Zhangetal.,2012),theprotoplastswereresuspendedinW5solutionandkeptat25°Cinthe

dark.All samples2days later, the transformationefficiencywasdeterminedby counting the

YFP-positivecellsusing imageanalysis,genomicDNAwas isolatedandusedasatemplatefor

thedeepsequencinglibrarypreparation,asdescribedin(Čermáketal.,2015).Briefly,40ngof

protoplastgenomicDNAwasusedastemplatetoamplifya354-bpregionoftheARF8A locus

encompassing the gRNA9 and gRNA10 target sites with primers TC324F and TC324R

(Supplemental Table 6), which have overhangs complementary to Nextera XT indices. PCR

productswerepurifiedwithAgencourtAMPureXPBeads(BeckmanCoulter,Brea,USA)and5

μlofthepurifiedPCRproductwasusedastemplateforthesecondPCRtoattachdualindices

and Illumina sequencing adapters. PCR products were purified with Agencourt AMPure XP

Beads, quantified and pooled. The pooled libraries were enriched for fragment sizes in the

range250 -700bpusingBluePippin (SageScience,Beverly,USA)and sequencedon Illumina

MiSeq platform (ACGT, Inc., Wheeling, USA). An average of 140,000 reads per sample was

generated (Supplemental Table 1, http://www.ebi.ac.uk/ena/data/view/PRJEB13819).

Sequencedataanalysiswasdoneasdescribedpreviously(Čermáketal.,2015),exceptdifferent

sequence variants were manually sorted into specific groups. All data are presented as the

35

mean of three biological replicates (three separate pools of protoplasts) plus/minus the

standarderror.Totestforthesignificanceofthedifferencebetweenthesamples,Tukeytest

formultiplecomparisonofmeansinR(version3.2.4RC)wasused(SupplementalTable4).

Testingmulti-gRNAexpressionsystemsintomatoprotoplastsbyqPCR

The Csy4, tRNA and ribozyme arrays of eight gRNAs (Supplemental Table 4) were created

directlyinprotoplastvectorspDIRECT_10C(Csy4)andpDIRECT_10E(tRNAandribozyme)using

Protocol3A, 3B and3C (SupplementalMethods), resulting inpTC634 (Csy4, gRNAorder49-

56),pTC635(Csy4,gRNAorder56-49),pTC636(tRNA,gRNAorder49-56),pTC637(tRNA,gRNA

order56-49),pTC638(ribozyme,gRNAorder49-56)andpTC639(ribozyme,gRNAorder56-49).

To construct the vector with eight gRNAs each expressed from an individual AtU6 (Pol III)

promoter, four AtU6:gRNA cassettes were first assembled into each pMOD_B2203 and

pMOD_C2200(resultinginpTC643andpTC644)usingProtocol3,exceptthefirst,lastandeach

reverse primer were re-designed to bind the AtU6 sequence. The AtU6 cassettes were

amplifiedfromBanI-digestedpTC363(firstcassette),aHindIIIandSgrAIfragmentfrompTC363

(intermediarycassettes)andaBsaHIfragmentfrompTC363(lastcassette).The35Sterminator

wasremovedfrompTC644bySnaBI/Eco53kI-mediatedreleaseoftheterminatorfragmentand

religationofthebackbone,resultinginpTC645.pTC643andpTC645wereassembledalongwith

pMOD_A0101 into thenon-T-DNAvectorpTRANS_100usingProtocol5, resulting in the final

vectorpTC646.

The CRISPR/Cas9 constructs were transformed into tomato protoplasts as described

above.PurifiedprotoplastDNAwasextracted48hourslaterandusedwithLunaUniversalqPCR

36

Master Mix (NEB) following the manufacturer’s protocol. All samples were run in technical

triplicateoftwobiologicalreplicates(twoseparatepoolsofprotoplasts)ina384-wellformaton

a LightCycler 480 Instrument (Roche). All qPCR primers are listed in Supplemental Table 6.

Primers for the genomic locus SGN-U346908 control were previously described (Expósito-

Rodríguezetal.,2008).Alldataarepresentedasthemeanplus/minusthestandarderrorwith

eachsamplerelativetothelevelsoftotalDNAasmeasuredbythegenomicslocuscontrol.

Geneeditingintomato,wheatandbarleyprotoplasts

TobuildthetomatoconstructpTC362withtwelvegRNAstargetingsixgenes,twoarraysofsix

gRNAs were cloned into pMOD_B2203 (with CmYLCV promoter) and pMOD_C2200 using

Protocol 3S1 and the two arrays were assembled together with the Csy4-Cas9 module

pMOD_A0501 into the non-T-DNA transformation backbone pTRANS_100 using Protocol 5

(SupplementalMethods).ThewheatvectorpJG612wasbuiltaccordinglyusingtheversionof

Cas9optimizedformonocotsinpMOD_A1510,asinglearrayofsixgRNAsdrivenbythePvUbi1

promoter in pMOD_B2112 and an empty module pMOD_C0000. Similarly, pMOD_A1510,

pMOD_B 2112 (PvUbi1) or pMOD_B2103 (CmYLCV), and the empty pMOD_C0000 modules

along with the transformation backbone pTRANS_100 were used to construct the barley

vectors expressing two gRNAs from the PvUbi1 (pTC441) or CmYLCV (pTC437) promoters,

targeting for deletion theMLO gene. Tomato protoplasts were isolated and transformed as

described above. Wheat protoplasts were isolated from ~10 day old plantlets of the bread

wheatcv.Bobwhite208.Seedsweresurfacesterilizedbyrinsingin70%ethanolfor10minand

soakingfor30minina1%sodiumhypochloritesolution,andasepticallygrowninMSmedium

37

at24°Cundera16-hlightcycle.Approximately20plantletswereharvestedineachexperiment,

cutinto~1-mmstripswitharazorblade,anddigestedwithanenzymesolution(1.5%cellulase

R10, 0.75%macerozyme R10, 0.6Mmannitol, 10mMMES, 10mMCaCl2, and 0.1% bovine

serumalbumin,pH5.7). The remaining stepswere identical as in theprotocol forprotoplast

isolation from tomato described above. Barley protoplasts were isolated from cv. Golden

Promiseusingthesameprotocol,exceptleavesfrom9-to14-day-oldplantlets(22°Cwith16h

light cycle) were used. The CRISPR/Cas9 constructs were co-transformed into ~200,000 cells

alongwiththeGFPexpressionplasmidpMOD_A3010theprotoplastswereresuspendedinW5

solutionandkeptat25°Cinthedark.GenomicDNAwasisolated2daysaftertransformation,

targeted deletions were detected using primers listed in Supplemental Table 6, cloned and

sequenced.

GeneeditingwithTrex2

Individual gRNAs were cloned into pMOD_B2515 (AtU6 promoter, for tomato) and

pMOD_B2518 (TaU6promoter, for barley) usingProtocol 2A and assembledwith 35S:Trex2-

P2A-AtCas9 in pMOD_A0901 (for tomato) or ZmUbi:Trex2-P2A-TaCas9 in pMOD_A1910 (for

barley) and the empty module pMOD_C0000 into the protoplast transformation backbone

pTRANS_100 to createpTC391 (gRNA23, tomatoANT1),pTC393 (gRNA24, tomatoANT1) and

pTC436 (gRNA38, barleyMLO) using PROTOCOL 5. The control plasmids pTC392 (gRNA23,

tomato ANT1), pTC394 (gRNA24, tomato ANT1) and pTC440 (gRNA38, barleyMLO) without

Trex2wereconstructedaccordingly,except35S:AtCas9 inpMOD_A0101andZmUbi:TaCas9 in

pMOD_A1110 were used instead of the Trex2 containing modules. The constructs were

38

transformed into tomato and barley protoplasts as described above and genomic DNA was

extracted2dayslater.TheANT1andMLOlociwereamplifiedbyPCR(seeSupplementalTable

6forprimers)andthegeneeditingratesweredeterminedusingT7endonucleaseIaccordingto

the manufacturer’s protocol (NEB, Ipswich, USA). Gel images were analyzed using ImageJ

(Schneider et al., 2012). In addition, theANT1 PCR productswere cloned and sequenced to

determine both frequency and type of mutations. For barley, the PCR products were first

digestedwith NcoI to enrich formutations and only the digestion-resistant product was gel

purified,clonedandsequenced.

GenetargetingwithCas9nickasesintobaccoandwheat

IndividualgRNAspacerswereclonedinmoduleB(pMOD_B2515fortobaccoorpMOD_B2518

forwheat)orC(pMOD_C2516fortobaccoorpMOD_C2518forwheat),downstreamofanAtU6

andAt7SLor twoTaU6promoters.TheGTdonorswere inserted into the resultingmoduleC

plasmids usingProtocol 4 (SupplementalMethods). A promoter-less T2A-gfp-nos terminator

cassette with 747 and 773 bp long flanking homology arms, and a 2530 bp long functional

gus:nptII gene fusion were used as donor templates for wheat and tobacco, respectively.

Finally,theBandCmoduleswereassembledwithselectedmoduleACas9variants(nuclease,

D10Anickase,H840nickaseordeadCas9expressedfromeither35SorZmUbipromoterintoa

BeYDV (pTRANS_201) or WDV (pTRANS_203) replicon vectors and used for A. tumefaciens

infiltrationoftobaccoleaves(pJG363,pJG365,pJG367,pJG369,pJG370,pJG380andpJG382)or

biolistic bombardment ofwheat scutella (pJG455, pJG456, pJG480, pJG481, pJG484, pJG486,

pJG488 and pJG493). Note that plasmids pJG455-pJG493 above used the octopine synthase

39

(OCS)terminatorsequenceinsteadofthemoreefficientHSPterminatorusedinthepublished

modules. The TaCas9 coding sequence and the rest of the plasmidswere identical with the

newermodulesincludedinthecurrentvectorset.Fourdifferenttransgeniclines(Wrightetal.,

2005)wereused todeterminegene targeting frequencies in tobaccoaspreviouslydescribed

(Baltesetal.,2014),withminormodifications.Leavesfrom1to6weeksoldplantswereused

forAgrobacteriumleafinfiltration.Foreachleaf,onehalfwasinfiltratedwithacontrolplasmid

pLSLZ.D.R(Baltesetal.,2014)thatservedasareferencetocontrolfortransformationefficiency

andtheotherhalfwithoneoftheCRISPR/Cas9constructs.Fourtosix leaveswere infiltrated

witheachconstructineachexperiment.Fivedaysafterinfiltration,theleaftissuewasstained

in the X-Gluc solution. Whole leaves were scanned and the stained area was quantified by

imageanalysisusingImageJ(Schneideretal.,2012).TheGTfrequencieswerenormalizedtothe

pLSLZ.D.RcontrolanddisplayedrelativetotheCas9nucleaseconstruct.Formolecularanalysis,

genomicDNAwasextracted fromtwo infiltrated leavespersample (three leafpuncheswere

takenfromeachleafandpooled)andusedasatemplateforPCRtoamplifytheleftandright

recombinationjunctionsusingprimerslistedinSupplementalTable6.PCRproductsfromboth

leaf sampleswere cloned and sequenced. To determineGT frequencies inwheat, immature

wheatscutella(0.5-1.5mm)wereisolatedfromprimarytillersharvested16daysafteranthesis

and used for biolistic transformation. Scutella isolation and culture conditions were as

describedinGil-Humanesetal.(2011).BiolisticbombardmentwascarriedoutusingaPDS-1000

gene gun. Equimolar amounts (1 pmol DNA mg-1 of gold) of plasmid were used for each

experimentwith60μgofgoldparticles(0.6μmdiameter)pershot.GFPimagesoftransformed

40

tissue were taken 7 days after transformation and GFP positive cells were counted. The GT

frequencieswerenormalizedtotheCas9nucleaseconstruct.

Accessionnumbers

ThedeepsequencingdataisavailableundertheEuropeanNucleotideArchive(ENA)accession

PRJEB13819(http://www.ebi.ac.uk/ena/data/view/PRJEB13819).

AllvectorsarepubliclyavailablefromAddgene(plasmid#90997–91225).AnnotatedvectorsequencesareavailablefromAddgeneandfromhttp://cfans-pmorrell.oit.umn.edu/CRISPR_Multiplex/

SUPPLEMENTALDATA

SupplementalFigure1.Universalarchitectureofexpressioncassetteswithuniquerestriction

sitesshown.

Supplemental Figure 2. GFP expression mediated by different promoter/terminator

combinationsintomatoprotoplasts.

SupplementalFigure3.FrequenciesofmutationsinducedusingdifferentsystemsforgRNA

expressionintomatoprotoplasts.

SupplementalFigure4.ShortindelsequencevariantsdetectedatgRNAsite9.

SupplementalFigure5.ShortindelsequencevariantsdetectedatgRNAsite10.

SupplementalFigure6.ShortindelsequencevariantsdetectedatbothgRNAsites9and10.

SupplementalFigure7.InsertionsfromtheARF8AlocusmappedtothevectorpTC364.

SupplementalFigure8.LongdeletionsequencevariantsdetectedbetweengRNAsites9and

10.

SupplementalFigure9.GFPexpressionmediatedbydifferentpromotersinwheatscutella.

41

SupplementalFigure10.MultiplexedtargetedmutagenesisinMedicagotruncatulausingCas9

andCsy4.

SupplementalFigure11.AnalysisofT1progenyfromfourMedicagotruncatulaT0plantswith

mutationsinNCR53,NCR54andNCR55.

SupplementalFigure12.SequencesofinsertionsdetectedattheANT1site#2intomato

protoplastsampleswithoutTrex2expression.

SupplementalFigure13.Detectionofgenetargetingintobaccoleaves.

SupplementalFigure14.GenetargetingwithCas9nickasesandgeminivirusreplicons(GVRs)in

wheat.

SupplementalTable1.Sequencingandreadfilteringdata.

SupplementalTable2.FrequenciesofindividualmutationtypesamongallARF8Asequencing

reads.

SupplementalTable3.MultipleTukeytestresults.

SupplementalTable4.SequencesofthegRNAsitesusedintheqPCRexperiment.

SupplementalTable5.Genotypesof46MedicagotruncatulahygromycinresistantT0plants

transformedwithaCas9Csy4constructexpressingsixgRNAstargetingthreegenes.

SupplementalTable6.Listofprimersusedinthisstudy.

SupplementalMethods.Protocolsforvectorassembly.

SupplementalDataSet1.Listofvectorsincludedinthetoolset.

SupplementalDataSet2.SequencesofNCR53,NCR54andNCR55locitargetedfordeletionin

46MedicagotruncatulaT0plants.

42

ACKNOWLEDGEMENTS

WethankmembersoftheVoytaslabforhelpfuldiscussionsandinsights.Weparticularlythank

LynnHufortechnicalassistance,LázaroPeres(UniversidadedeSãoPaulo,Brazil)andAgustin

Zsögön (Universidade Federal de Viçosa, Brazil) for input on target genes in tomato, Joseph

Guhlin (College of Biological Sciences, University of Minnesota) for input onMedicago NCR

target genes, Aaron Hummel for providing the plasmids with structurally optimized gRNA

scaffolds, Evan Ellison for the GmUbi promoter reagents, and Kit Leffler for help with the

figures. FundingwasprovidedbygrantsfromtheNationalScienceFoundation (IOS-1339209

and IOS-1444511) and the Department of Energy (DE-SC0008769).

AUTHORS’CONTRIBUTIONS

TCdesignedthevectorsystemandworkplan.TC,JG,CGSandRLGconstructedthevectors.TC

performedtheexperimentsintomatoprotoplasts.SJCperformedtheexperimentsinMedicago

truncatula.JGperformedtheexperimentsinwheatprotoplastsandgenetargetingexperiments

intobaccoandwheat.EKperformedtheexperimentsinbarleyprotoplastsandcarriedoutthe

molecularanalysisof thegenetargetingevents in tobacco.RCanalyzedthedeepsequencing

data.TJYKdesignedandbuiltthesetofonlinetools.JJBperformedtheqPCRanalysistoassess

deletionfrequencies.JWMperformedtheanalysisofGUSstainingintobaccoleaves.TC,JG,SC

andDVwrotethemanuscript.

57

NumberofT0plants(outof46)

Targetgene

DeletionbetweengRNAsites

InversionbetweengRNAsites

IndelatgRNAsite#1only

IndelatgRNAsite#2only

IndelsatbothgRNAsites

Totalmutant

NCR53(multicopy) 17 4 11 1 23 43NCR54 0 0 0 8 0 8NCR55 7 0 5 0 1 12

Table1.MultiplextargetedmutagenesisinMedicagotruncatulausingCas9andCsy4.

Golden Gate assembly using Type IIs enzymes

CCDB

ORClone gRNA(SINGLE STEP)pF U

S_A

pF U

S_B

pL

R

Assemble a TALEN(THREE STEPS)

Transforma

tion Backbone (TB)

A BGolden Gate assembly using Aarl

CCDB

Transforma

tion Backbone (TB)

A

B

C

CCTG

CTAG

GATC

GGCCGGAC

CCGG TCAC

AGTG

A B C

Aarl AarlAarl Aarl Aarl Aarl Aarl Aarl

DIRECT CLONING VECTORS MODULAR ASSEMBLY VECTORS

Figure 1. Two sets of vectors for direct cloning or modular assembly of genome engineering reagents. A) Direct cloning vectors were designed to speed-up the cloning process. Specificity determining elements (gRNAs or TAL repeats) are cloned directly into the transformation backbone (e.g. T-DNA). B) The modular assembly vectors enable combination of different functional elements. Specificity determining elements (gRNAs, TAL repeatsand donor templates for gene targeting) are first cloned into intermediate module vectors. Custom-selected modules are then assembled together intothe transformation backbone (e.g. T-DNA).

A

C

TGAATGTCATAAACACTCATGAATTTTGGCCTCATCAGTTTGTGATGGAAAA

TGAATGTCATAAACACTCATGAAT-----------------GTGATGGAAAA: C1TGAATGTCATAAACACTCATGAATTT--GCCTCATCAGTTTGTGATGGAAAA: C2TGAATGTCATAAACACTCATGAAT----GCCTCATCAGTTTGTGATGGAAAA: C3TGAATGTCATAAACACTCATGAATTTTG-CCTCATCAGTTTGTGATGGAAAA: C4

WPT52-4-4 - Medtr4g020620

ATTTAGTTCCTGTGAATGTCATAAACACTCATGAATTTTGGCCTCATCAGTTTGTGATGGAAAAAG

A---------------------------------------------------------------AG

WPT52-4-4 - Medtr2g013650

4g020620

2g013650

bar

ACTIN

WPT

52-4

-1W

PT52

-4-2

WPT

52-4

-3W

PT52

-4-4

WPT

52-4

-5W

PT52

-4-6

WPT

52-4

-7W

PT52

-4-8

HM34

0 (D

)HM

340

(UD)

B

TGTCATAAACACTCATGAATTTTGGCCTCATCAGTTTGTGATGGA

Medtr4g020620

4g020620 F

4g020620 R

2g013650 R

2g013650 F

Medtr2g013650

Figure 2. TALEN-mediated mutagenesis in Medicago truncatula using direct cloning vectors. A) Maps of two Medicago genes targeted for mutagenesis using a single TALEN pair. PCR primers used for screening in panel B are shown as green arrowheads. The sequence of the TALEN target site is shown with TALEN binding sites underlined. B) HaeIII PCR digestion screening of eight T1 progeny of plant WPT52-4. The amplified locus is indicated on the right. bar and ACTIN are controls that amplify the transgene and a native gene, respectively. HM340 (D), WT product digested with HaeIII; HM340 (UD), undigested WT product. C) DNA sequences of undigested amplicons from plant WPT52-4-4. The reference sequence of the unmodified locus is shown on the top. TALEN binding sites are underlined. HaeIII restriction site used for the screening is in bold red text.

50

40

30

20

10

0

AtU6+

At7SL

Impro

ved g

RNA

AtU6+

AtU6

Csy4

tRNA

Riboz

yme

Non-t

ransfo

rmed

ALL MUTATIONSC

B

A

CCCTCAAAGATCAGGCTGGCAGC...62 bp...CCTAATTAACCAGAAAACTTAC

Unmodified

Long deletion

Short indels at gRNA9 site

Short indels at gRNA10 site

Short indels at both sites

Inversion

Long deletion plus insertion

gRNA9 gRNA1085bp

5’ 3’

Figure 3. Comparison of different systems for expressing multiple gRNAs. A) Structure of six constructs for expressing gRNA9 and gRNA10, which both target the tomato ARF8A gene. CmYLCV, cestrum yellow leaf curling virus promoter; Csy4, 20 bp Csy4 hairpin; tRNA, 77 bp pre-tRNAGly gene; rb, 15 bp ribozyme cleavage site; ribozyme, 58 bp synthetic ribozyme; AtU6, Arabidopsis U6 promoter; At7SL Arabidopsis 7SL promoter. B) Possible editing outcomes due to expression of both gRNAs. Sequences targeted by gRNA9 and gRNA10 are shown on the top Cleavage sites are indicated by arrows. C) Overall mutation frequencies as determined by deep sequencing. Error bars represent SE of three replicates (pools of protoplasts). Statistical significance was determined by Tukey’s test. *P < 0.05 and ***P < 0.001.

*Improved gRNA scaffold

Csy4 Csy4 Csy4CmYLCV gRNA9 gRNA10 35S polyA

CmYLCV gRNA9 gRNA10 35S polyAtRNA tRNAtRNA

rb ribozymeCmYLCV gRNA9 rb gRNA10 35S polyA

AtU6 gRNA9 AtU6 gRNA10 TTTTTTTTTTTT

gRNA9 At7SL gRNA10 TTTTTTTTTTTTAtU6

gRNA9 At7SL gRNA10 TTTTTTTTTTTTAtU6

Ove

rall

Mut

atio

n Fr

eque

ncy

C

Figure 4. Evaluation of eight gRNAs in various polycistronic expression systems and the effect of position in the array on gRNA activity. A) Structure of seven constructs for expressing gRNAs 49 -56 targeting four tomato genes for deletion. B) Detection of deletions between gRNA sites using qPCR. Each gene is targeted for deletion with two gRNAs designed to create a 3 kb deletion to prevent amplification of the non-deleted, WT template. Deletion frequency wsa quantified by qPCR using primers shown as red and blue arrowheads. C) Deletion frequencies in tomato protoplasts as measured by qPCR. Positions of respective gRNAs in the array are specified for each construct. Error bars represent SE of two replicates.

ACm

YLCV

gRNA

49

gRNA

50

35S p

olyA

gRNA

51

gRNA

52

Csy4

Csy4

Csy4

Csy4

Csy4

Csy4

Csy4

Csy4

Csy4

gRNA

53

gRNA

54

gRNA

55

gRNA

56

CmYL

CV

gRNA

56

gRNA

55

35S p

olyA

gRNA

54

gRNA

53

Csy4

Csy4

Csy4

Csy4

Csy4

Csy4

Csy4

Csy4

Csy4

gRNA

52

gRNA

51

gRNA

50

gRNA

49

CmYL

CV

35S p

olyA

tRNA

Gly

gRNA

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gRNA

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gRNA

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gRNA

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gRNA

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tRNA

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gRNA

56

gRNA

55

gRNA

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gRNA

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gRNA

52

gRNA

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gRNA

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gRNA

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CmYL

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35S p

olyA

tRNA

Gly

tRNA

Gly

tRNA

Gly

tRNA

Gly

tRNA

Gly

tRNA

Gly

tRNA

Gly

tRNA

Gly

tRNA

Gly

rb rb rb rb rb rb rb rbCmYL

CV

gRNA

49

gRNA

50

35S p

olyA

gRNA

51

gRNA

52

riboz

yme

gRNA

53

gRNA

54

gRNA

55

gRNA

56

CmYL

CV

gRNA

56

gRNA

55

35S p

olyA

gRNA

54

gRNA

53

rb rb rb rb rb rb rb rbgRNA

52

gRNA

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gRNA

50

gRNA

49

riboz

yme

gRNA

49

gRNA

50

gRNA

51

gRNA

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gRNA

53

gRNA

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gRNA

55

gRNA

56

AtU6

TTTT

TT

TTTT

TT

TTTT

TT

TTTT

TT

TTTT

TT

TTTT

TT

TTTT

TT

TTTT

TT

AtU6

AtU6

AtU6

AtU6

AtU6

AtU6

AtU6

BWT locus

3 kb - no amplification

Deleted locus

100-200 bp - amplifies in qPCR

Del

etio

n Fr

eque

ncy

Solyc02g077390 - gRNAs 49/50

Csy4

- 1/2

Csy4

- 7/8

tRNA

- 1/2

tRNA

- 7/8

Ribo -

1/2

Ribo -

7/8 U6

0

0.2

0.4

0.6

0.8

1.0

1.2Solyc06g074240 - gRNAs 55/56

Del

etio

n Fr

eque

ncy

Csy4

- 7/8

Csy4

- 1/2

tRNA

- 7/8

tRNA

- 1/2

Ribo -

7/8

Ribo -

1/2 U6

0

0.2

0.4

0.6

0.8

1.0

1.2Solyc02g090730 - gRNAs 53/54

Del

etio

n Fr

eque

ncy

Csy4

- 5/6

Csy4

- 3/4

tRNA

- 5/6

tRNA

- 3/4

Ribo -

5/6

Ribo -

3/4 U6

0

0.2

0.4

0.6

0.8

1.0

1.2

C

A1930 bp (1306 bp)Solyc06g074350

gRNA11 gRNA12

1510 bp (409 bp)Solyc02g085500

gRNA13 gRNA14

670 bp (388 bp)Solyc02g090730

gRNA15 gRNA16

1497 bp (684 bp)Solyc06g074240

gRNA17 gRNA18

1933 bp (1536 bp)Solyc02g077390

gRNA19 gRNA20

1551 bp (313 bp)Solyc11g071810

gRNA21 gRNA22

Solyc06g074350AGATCATGATAGATCCAGATGTTCCTGGTCCTAGTGATCCATATCT // AGAGGATTTGACCATCTTTACAGGATTGTCACAGACATTCCAGGCACTAGATCATGATAGATCCAG---------------------------- // -------------------------------------------GCACTAGATCATGATAGATCCAG---------------------------- // -------------------------------------------GCACTAGATCATGATAGATCCAG---------------------------- // -------------------------------------------GCACT

gRNA11 gRNA12

Solyc02g085500TAGTTCCTTCGTGTCTGAAGAAGAAGAATGTGAAGAGGAGATCAATTT // AGTGAAGAAATCTCAGGACCCGTACTAAGATTTCAAGAGATCGATGTAGTTCCTTCG------------------------------------- // -------------------------GAAGATTTCAAGAGATCGATGTAGTTCCTTCG------------------------------------- // -----------------------------ATTTCAAGAGATCGATG

gRNA13 gRNA14

Solyc02g090730ATGTTCCTCCTCACTATGTATCTGCCCCCGGCACCACCACGGCGCGGT // TTAGCATGTGGGAGTAGAGGTGCATTATATTGTTTGCTGGGATTGAATGTTCCTCCT------------------------------------- // -----------------------------------GCTGGGATTGAATGTTCCTCCT------------------------------------- // -----------------------------------GCTGGGATTGAATGTTCCTCCTC------------------------------------ // ----------------------------------TGCTGGGATTGA

gRNA15 gRNA16

Solyc11g071810AAATCCCTTCGATTCGTCGTAAGTACTCTCTCTATCTCTCTTAATAAA // GAGAAAAGACAACGTGTTCCTTCTGCGTACAACCGATTCATCAAGTAAATCCCTTCGA------------------------------------ // ------------------------GCGTACAACCGATTCATCAAGTAAATCCCTTCGA------------------------------------ // ----------------------------ACAACCGATTCATCAAGTAAATCCCTTCG------------------------------------- // -------------------------CGTACAACCGATTCATCAAGT

gRNA21 gRNA22

Solyc06g074240GATCATTATCGGAGCTGGCCCTGCTGGGCTCAGGCTAGCTGAACAAGT // CTATTGGTGGGAATTCAGGGATAGTTCATCCATCAACAGGGTA