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Precise and efficient genome editing in Klebsiella pneumoniae using 1 CRISPR-Cas9 and CRISPR-assisted cytidine deaminase 2 Yu Wang 1 , Shanshan Wang 2 , Weizhong Chen 1 , Liqiang Song 1 , Yifei Zhang 1 , Zhen 3 Shen 3 , Fangyou Yu 4 , Min Li 3 , and Quanjiang Ji 1* 4 5 1 School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China. 6 2 Department of Laboratory Medicine, Wenzhou Medical University, Wenzhou 325000, China. 7 3 Department of Laboratory Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong 8 University, Shanghai, 200127, China. 9 4 Department of Clinical Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji 10 University, Shanghai 200443, China. 11 12 13 * Corresponding author: Quanjiang Ji; E-mail: [email protected] 14 15 AEM Accepted Manuscript Posted Online 14 September 2018 Appl. Environ. Microbiol. doi:10.1128/AEM.01834-18 Copyright © 2018 American Society for Microbiology. All Rights Reserved. on July 22, 2020 by guest http://aem.asm.org/ Downloaded from

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Page 1: Precise and efficient genome editing in Klebsiella pneumoniae using CRISPR … · 106 In this study, we devel oped a CRISPR -Cas9 -mediated genome editing method as well as a 107

Precise and efficient genome editing in Klebsiella pneumoniae using 1

CRISPR-Cas9 and CRISPR-assisted cytidine deaminase 2

Yu Wang1, Shanshan Wang

2, Weizhong Chen

1, Liqiang Song

1, Yifei Zhang

1, Zhen 3

Shen3, Fangyou Yu

4, Min Li

3, and Quanjiang Ji

1* 4

5

1School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China. 6

2Department of Laboratory Medicine, Wenzhou Medical University, Wenzhou 325000, China. 7

3Department of Laboratory Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong 8

University, Shanghai, 200127, China. 9

4Department of Clinical Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji 10

University, Shanghai 200443, China. 11

12

13

*Corresponding author: Quanjiang Ji; E-mail: [email protected] 14

15

AEM Accepted Manuscript Posted Online 14 September 2018Appl. Environ. Microbiol. doi:10.1128/AEM.01834-18Copyright © 2018 American Society for Microbiology. All Rights Reserved.

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ABSTRACT 16

Klebsiella pneumoniae is a promising industrial microorganism as well as a major human 17

pathogen. The recent emergence of carbapenem-resistant K. pneumoniae has posed a serious 18

threat to public health worldwide, emphasizing a dire need for novel therapeutic means against 19

drug-resistant K. pneumoniae. Despite the critical importance of genetics in bioengineering, 20

physiology study, and therapeutic-means development, genome editing, in particular, the highly 21

desirable scarless genetic manipulation in K. pneumoniae is often time-consuming and laborious. 22

Here we report a two-plasmid system pCasKP/pSGKP for precise and iterative genome editing in 23

K. pneumoniae. By harnessing the CRISPR-Cas9 genome cleavage system and the lambda-Red 24

recombination system, pCasKP/pSGKP enabled highly efficient genome editing in K. pneumoniae 25

using a short repair template. Moreover, we developed a cytidine base editing system pBECKP for 26

precise C→T conversion in both the chromosomal and plasmid-borne genes by engineering the 27

fusion of a cytidine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP/pSGKP 28

and the pBECKP tools, the blaKPC-2 gene was confirmed to be the major factor that contributed to 29

the carbapenem resistance of a hypermucoviscous carbapenem-resistant K. pneumoniae strain. 30

The development of the two editing tools will significantly facilitate the genetic engineering of K. 31

pneumoniae. 32

33

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IMPORTANCE 34

Genetics is a key means to study bacterial physiology. However, the highly desirable scarless 35

genetic manipulation is often time-consuming and laborious for the major human pathogen K. 36

pneumoniae. We developed a CRISPR-Cas9-mediated genome editing method as well as a 37

cytidine base editing system, enabling rapid, highly efficient, and iterative genome editing in both 38

industrial and clinically isolated K. pneumoniae strains. We applied both the tools in dissecting the 39

drug-resistant mechanism of a hypermucoviscous carbapenem-resistant K. pneumoniae, 40

elucidating that the blaKPC-2 gene was the major factor that contributed to the carbapenem 41

resistance of the hypermucoviscous carbapenem-resistant K. pneumoniae strain. The utilization of 42

the two tools will dramatically accelerate a wide variety of investigations in diverse K. 43

pneumoniae strains and relevant enterobacteriaceae species, such as gene characterization, drug 44

discovery, and metabolic engineering. 45

46

47

KEYWORDS 48

CRISPR, Cas9, Klebsiella pneumoniae, genetic engineering, genome editing, base editing 49

50

51

52

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INTRODUCTION 53

Klebsiella pneumoniae is a high-GC content, Gram-negative bacillus of the 54

Enterobacteriaceae family, widely distributed in the natural environment and on mucosal surfaces 55

of mammals. It is considered as a promising industrial microorganism, because of its capacity in 56

naturally synthesizing a diverse range of valuable chemicals (1). In addition, K. pneumoniae is a 57

major human pathogen, causing a wide variety of hospital- and community-acquired infections, 58

such as pneumoniae, bacteremia and urinary tract infections (2). In recent years, the emergence of 59

hypermucoviscous and multidrug-resistant K. pneumoniae, in particular, the carbapenem-resistant 60

hypermucoviscous K. pneumoniae, has posed a severe public crisis worldwide (3-5). Thereby, 61

novel therapeutic means against multidrug-resistant K. pneumoniae infections are urgently needed. 62

The development of novel therapeutic means against drug-resistant K. penumonia would 63

benefit greatly from efficient and convenient genome editing and screening tools, which allow 64

effective identification of key genes and pathways responsible for bacterial virulence and drug 65

resistance. Although the lambda-Red recombination system has been developed and widely 66

utilized for genetic manipulation and the CRISPRi system has been developed recently for 67

transcriptional inhibition in K. pneumoniae, the highly desirable scarless and precise genome 68

editing in K. pneumoniae is still time-consuming and laborious (6, 7). For instance, to construct a 69

markerless deletion mutant in K. pneumoniae, a target gene is first replaced by an antibiotic 70

marker via a double-crossover homologous-recombination process mediated by the lambda-Red 71

recombination proteins (Gam, Bet, and Exo). Second, the antibiotic marker is eliminated by the 72

utilization of a helper plasmid expressing the FLP recombinase. The FLP recombinase directly 73

binds to the repeated FLP recognition sites flanking the antibiotic gene and catalyzes the 74

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elimination reaction, leaving an FRT scar in the place of the target gene. When multiple rounds of 75

genetic modification are performed using the aforementioned method, the introduction of multiple 76

FRT scars in the genome may lead to genome instability by causing genome rearrangement (8). 77

The recent discovered CRISPR-Cas9 system allows for the efficient generation of a 78

double-strand break (DSB) at a desired site of the target genome (9, 10), thereby raising the 79

possibility of one-step scarless genome editing in K. pneumoniae. The CRISPR system is an 80

adaptive immune system and is utilized by bacteria and archaea to fight against invading phages 81

and foreign plasmids (11, 12). The wildly utilized CRISPR-Cas9 system is composed of two 82

components: the Cas9 nuclease from Streptococcus pyogenes and a single artificial chimeric guide 83

RNA (sgRNA) (13). The sgRNA directs the Cas9 protein to a target genomic locus through 84

complementary base-pairing to a target sequence in the presence of a downstream 5’-NGG-3’ 85

protospacer adjacent motif (PAM) (14). After that, the Cas9 nuclease creates a DSB within the 86

base-pairing region (13). Given the lack of the non-homologous end joining (NHEJ) pathway in 87

most bacteria including Klebsiella pneumoniae, chromosomal cleavage is lethal to bacterial cells 88

unless it is repaired by the homologous recombination (HR) pathway with the utilization of 89

exogenously supplied donor DNA repair templates (15). Thereby, precise genetic manipulation, 90

including gene deletions, point mutations, and gene insertions can be achieved by simply 91

customizing an approximately 20 nucleotide (nt) spacer sequence and a designed donor repair 92

template. 93

Furthermore, the recent developed CRISPR RNA-guided deaminase systems enable precise 94

base editing, opening a new avenue for genome editing in biology. Until now, two kinds of base 95

editors have been developed: the cytidine editor BEC (16, 17) and the adenosine editor ABE (18). 96

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Each base editor is comprised of a Cas9 nickase (D10A in the case of SpCas9) or a dead Cas9 97

protein (D10A and H840A in the case of SpCas9) and a deaminase fused to the Cas9 protein. 98

Relying on the base-pairing between a target sequence and the 20 nt guide RNA sequence, the 99

tethered deaminase can be directed to any target locus to perform nucleosides deamination through 100

a deamination reaction (C→U for the BEC editor and A→I for the ABE editor). In living cells, the 101

DNA repair or replication mechanism would efficiently convert the U:G or I:T heteroduplex pairs 102

to the desired T:A or G:C pairs. Distinct from the CRISPR-Cas9-mediated genome editing, the 103

base editors directly catalyze the conversions of nucleosides without the formation of DSB or the 104

utilization of a donor template. 105

In this study, we developed a CRISPR-Cas9-mediated genome editing method as well as a 106

base editing system, enabling rapid, highly efficient, and iterative genome editing in both 107

industrial and clinically isolated K. pneumoniae strains. By using both the genome editing tools, 108

we confirmed that the blaKPC-2 gene was the major factor that contributed to the carbapenem 109

resistance of a hypermucoviscous carbapenem-resistant K. pneumoniae strain. The development of 110

the genome editing tools will dramatically accelerate a wide variety of investigations in K. 111

pneumoniae. 112

113

RESULTS 114

Establishment of a single-plasmid CRISPR-Cas9 system in K. pneumoniae. To develop a 115

convenient and scarless genetic manipulation method in K. pneumoniae, we first sought to harness 116

the CRISPR-Cas9 system for genome editing. To access the functionality of the CRISPR-Cas9 117

system in K. pneumoniae, we constructed a single-plasmid system pCas9-sgRNAKP that 118

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expressed both the well-studied Streptococcus pyogenes Cas9 protein and the sgRNA in the same 119

plasmid (19). The transformation of the empty pCas9_sgRNAKP plasmid into K. pneumoniae 120

yielded a lawn of colonies, whereas the transformation of the nonessential dhaF gene 121

spacer-introduced pCas9-sgRNAKP plasmid only produced a few colonies (Fig. 1), strongly 122

indicating the effective cleavage of bacterial genome by the CRISPR-Cas9 system. 123

Next, we assembled the repair templates (~ 1 kb each) of the dhaF gene into the 124

dhaF-spacer-introduced pCas9-sgRNAKP plasmid to test the functionality of the system for gene 125

deletion in K. pneumoniae. The transformation of the assembled plasmid into K. pneumoniae 126

yielded only fewer than 5 colonies (Fig. 1). Further PCR screening analysis revealed that none of 127

them were the desired deletion mutants, indicating that the intrinsic homologous recombination 128

capacity of K. pneumoniae was not great enough for the directly repair of the lethiferous 129

double-stranded DNA break of the genome. 130

To alleviate the toxicity of chromosomal cleavage by the Cas9 nuclease, two versions of 131

Cas9 nickase expression plasmids, pnCas9D10A_sgRNAKP and pnCas9H840A_sgRNAKP were 132

constructed by mutating the active sites of Cas9 protein Aps10 or His840 to Ala, respectively. The 133

transformations of the Cas9 nickase plasmids containing both the dhaF-spacer and the 134

corresponding repair template (~ 1 kb each) indeed yielded plenty of colonies. However, PCR 135

screening and further sequencing revealed that no desired homologous-recombination-repair 136

events were observed (Fig. S1). It is likely that K. pneumoniae preferred to accurately repair the 137

DNA nick using the complementary strand, rather than the exogenous donor templates. 138

Development of the two-plasmid system pCasKP/pSGKP for genome editing. Phage 139

recombination systems, such as lambda-Red and Rac-RecET, possess a stronger recombination 140

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capacity than that of normal bacterial cells (20, 21). We sought to increase the homologous 141

recombination capacity of K. pneumoniae by introducing the phage lambda-Red recombination 142

system into the bacteria. To achieve this, we designed and constructed a two-plasmid system 143

pCasKP/pSGKP (Fig. 2A-B). The pCasKP plasmid expressed the Cas9 protein under the control 144

of the constitutive K. pneumoniae rpsL promoter and the lambda-Red recombination proteins 145

(Gam, Bet and Exo) under the control of an L-arabinose-inducible ParaB promoter. The pSGKP 146

plasmid expressed the sgRNA under the control of the synthetic constitutive J23119 promoter (22). 147

Two reversed BsaI sites were inserted between the J23119 promoter and the sgRNA scaffold for 148

the seamless and one-step assembly of spacers (Fig. S2). In addition, the temperature-sensitive 149

replicon repA101ts (23) and the sucrose-sensitive gene sacB (24) were introduced into the pCasKP 150

and pSGKP plasmids, respectively, for easy plasmid curing after editing. 151

To assess the genome editing ability of the constructed two-plasmid system, we sought to 152

delete the dhaF gene in the industrial K. pneumoniae strain KP_1.6366. To do this, the 153

pCasKP-apr plasmid was first electroporated into the wild-type industrial K. pneumoniae strain 154

KP_1.6366 to obtain the pCasKP-apr-harboring strain. After the induction of L-arabinose, the 155

cells containing the pCasKP-apr plasmid were collected and prepared as the competent cells. Then, 156

the dhaF-deletion plasmid pSGKP-dhaF-HR was transformed into the aforementioned 157

pCasKP-apr-harboring competent cells by electroporation. The pSGKP-dhaF-HR plasmid was 158

constructed by assembling both the dhaF-spacer and the repair arms of the dhaF gene (~ 1 kb 159

each) into the pSGKP-km plasmid. The subsequent transformation yielded more than one 160

thousand colonies. Twenty colonies were randomly picked to test the editing efficiency. As shown 161

in Fig. 2C, the successful deletion of the dhaF gene was confirmed in all the picked colonies by 162

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both PCR and sequencing. 163

To simplify the plasmid construction procedures and accelerate the genome editing process, 164

we attempted to utilize the linear homologous DNA fragment as the repair templates. The 165

lambda-Red recombination system is capable of using the plasmid-borne donor DNA, linear 166

double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) as the repair templates (25). To 167

evaluate the editing efficiency of linear repair templates, a linear dsDNA (~ 500 bp each) and an 168

ssDNA (45 nt each) were co-transformed individually with the dhaK-spacer-introduced 169

pSGKP-km plasmid (pSGKP_dhaK) into the L-arabinose-induced pCasKP-apr-harboring cells to 170

delete the dhaK gene (26). The transformations of the pSGKP_dhaK plasmid (only dhaK spacer) 171

and the pSGKP_dhaK_HR plasmid (dhaK spacer and assembled with ~ 500 bp each repair 172

templates) were used as the negative and positive controls, respectively. As shown in Fig. 2D and 173

Fig. S3A, more than one thousand colonies were observed for all the transformations containing 174

any type of the donor repair templates, whereas less than 10 colonies were obtained for the 175

transformation without repair template. Further PCR screening and sequencing showed that the 176

deletion efficiencies were 100% for all the transformations containing the repair templates (Fig. 177

S3B-S3D). Moreover, we assessed the capacity of the two-plasmid system pCasKP/pSGKP for 178

deleting the fosA gene with the utilization of ssDNA as the repair template (27). As shown in Fig. 179

S4, the editing efficiency was 9/10. 180

In addition to gene deletion, the two-plasmid system pCasKP/pSGKP was used for gene 181

insertion in K. pneumoniae. We attempted to replace the fosA gene with the mcherry gene. We 182

co-transformed the fosA-spacer-introduced pSGKP-km plasmid (pSGKP-fosA) and the mcherry 183

gene with 45 bp homology extensions into the pCasKP-apr-harboring KP_1.6366 strain by 184

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electroporation (Fig. S5A). More than one hundred colonies were recovered and the insertion 185

efficiency was 9/10 (Fig. S5B). Together, these experiments demonstrated that the two-plasmid 186

pCasKP/pSGKP system possessed a great capacity for genetic manipulation in K. pneumoniae 187

with the utilization of a short repair template. 188

Complicate physiology study and metabolic engineering of K. pneumoniae requires the 189

genetic manipulation of multiple genes, thereby requiring multiple rounds of genome editing. For 190

the second-round editing, the spacer-incorporated pSGKP-km plasmid need to be recycled for 191

different target loci while the pCasKP-apr plasmid can be maintained to express the Cas9 protein 192

and the lambda-Red recombination system (Fig. 3). We inoculated one colony containing the 193

desired dhaK deletion into LB with the supplementation of apramycin. The cells were cultured at 194

30 °C overnight. Next, a fraction of the cells was streaked onto a LB agar plate containing 195

apramycin and sucrose, and incubated at 30 °C until colonies were visible. As shown in Fig. S6A, 196

all the four randomly picked colonies could only grow normally on the plate containing apramycin, 197

whereas none of them could grow on the plate containing both apramycin and kanamycin, 198

confirming the successful removal of the pSGKP-km plasmid with the maintenance of the 199

pCasKP–apr plasmid. Next, the dhaF gene was deleted in the pSGKP-dhaK-cured cells with the 200

efficiency of 10/10 (Fig. S6B). After finishing all the desired genome editing, both the 201

pCasKP-apr and the pSGKP-km plasmids could be easily cured by culturing the cells at 37 °C and 202

in the presence of sucrose (Fig. S6C). 203

To expand the utility of the two-plasmid system, we tested the editing efficiency of the 204

system in clinically isolated K. pneumoniae strains, KP_3744 and KP_5573. The editing 205

efficiencies of three different genes in the KP_3744 strain and four different genes in the KP_5573 206

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strain were systematically investigated. The deletion efficiencies of all the genes tested in the 207

KP_3744 strain (pyrF [Fig. 4A], fepB [Fig. 4B], and ramA [Fig. S7A]) and KP_5573 strain (fosA 208

[Fig. 4C], pyrF [Fig. S7B], fepB [Fig. S7C], and ramA [Fig. 4D]) were 100% (28-30). In addition 209

to PCR screening and sequencing, we used the growth defect assay and the fosfomycin-resistance 210

assay to verify the deletion of pyrF and fosA, respectively. The cells lacking the pyrF gene 211

(encoding orotidine 5-phosphate decarboxylase) have a growth defect in uracil-free synthetic 212

chemically defined medium (CDM) (31). The disruption of the fosA gene (encoding dimeric 213

Mn2+

- and K+-dependent glutathione S-transferase) renders the cells more susceptive to 214

fosfomycin. 215

Development of the single-plasmid system pBECKP for base editing. The 216

CRISPR-Cas9-mediated genome editing method generates a DSB and requires a donor repair 217

template for editing. We sought to further simplify the editing process by developing a base editor 218

in K. pneumoniae (Fig. 5A). The base editor directly mutates the target site without generating a 219

DSB or using a repair template. The cytidine base editor has the potential to inactivate genes via 220

converting four codons (CAA, CAG, CGA and TGG) into premature stop codons in a 221

programmable manner. To harness the cytidine base editor for base editing in K. pneumoniae, we 222

constructed a single-plasmid editing system pBECKP (Fig. 5B). The low-copy pBECKP plasmid 223

expressed the sgRNA under the control of the J23119 promoter and the fusion protein of Cas9 224

nickase (nSpCas9, D10A) and rAPOBEC1 deaminase with a 16-residue XTEN linker under the 225

control of a weak promoter (32). Two BsaI sites and the sacB gene were introduced into the 226

plasmid for convenient spacer assembly and plasmid curing, respectively. 227

To assess the capacity of the pBECKP system in base editing in K. pneumoniae, we 228

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transformed a fosA-spacer-introduced pBECKP-km plasmid into the clinically isolated KP_5573 229

strain. The fosA spacer contained a potentially editable “TC7C8” motif. The C→T conversions of 230

either or both the Cs at the positions of 7 and 8 could result in a premature stop codon in the fosA 231

gene. As shown in Fig. 5C, both the Cs at positions 7 and 8 were successfully mutated to Ts with 232

100% efficiencies in all the picked 8 colonies. The high base editing efficiency of the fosA gene 233

was also observed in the industrial KP_1.6366 strain (Fig. S8). In addition, we tested the 234

capability of the pBECKP system in editing C-rich regions in the genome. Two C-rich spacers 235

within the dhaK gene were assembled individually into the pBECKP-km plasmid. The plasmids 236

were transformed individually into the KP_1.6366 strain. As shown in Fig. 5D and Fig. S9, 237

various editing products with the conversions of Cs at different positions were obtained. These 238

results demonstrated that the pBECKP system could efficiently convert C to T in a variety of K. 239

pneumoniae strains. 240

The human BE3 base editor has a strong cytidine-deamination capacity within the mutational 241

spectra from positions 4-8 (termed activity window) (16). Within the activity window, the 242

base-editing system has a high C to T conversion efficiency. Outside the activity window, the base 243

editing could be detected occasionally, but the conversion efficiency reduced drastically. Because 244

the activity window of cytidine base editor may not be identical in different species, we 245

systematically examined the activity window and the sequence context preference of the pBECKP 246

system in K. pneumoniae. Ten distinct spacers containing Cs at different positions were assembled 247

into the pBECKP plasmid, respectively. The plasmids were transformed into KP_1.6366 strain, 248

respectively. As shown in Fig. 6, the editing efficiency of TC was higher than that of CC and AC. 249

GC had the lowest editing efficiency, consistent with the sequence context preference of the 250

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mammalian base editor (16). The Cs from the TC motif at positions 3 to 8 were converted to Ts 251

with the efficiencies of almost 100%, whereas the editing efficiencies of the Cs at other positions 252

were much lower, indicating the activity window of the pBECKP system was from positions 3 to 8. 253

Intriguingly, a few Cs at position 9 of the spacer 8 and position 7 of the spacer 10 were mutated to 254

As, but not Ts (Fig. 6). The editing byproduct was also observed in the editing process of 255

eukaryotic base editors (33-35). 256

Dissection of drug-resistant mechanisms using the two editing systems. The quick 257

dissemination of carbapenem-resistant K. pneumoniae has posed a severe threat to public health 258

worldwide. The mobile genetic elements encoding carbapenemases dramatically accelerate the 259

global expansion of carbapenem resistance. The acquirable carbapenemases are largely divided 260

into the KPC, NDM, OXA-48, VIM and IMP types (36). These carbapenemases are often 261

coproduced with extended-spectrum beta-lactamase (ESBL) in clinically isolated 262

carbapenem-resistant K. pneumoniae. We used both the pCasKP/pSGKP and the pBECKP systems 263

to verify the contribution of carbapenemases in carbapenem resistance. 264

First, we sought to delete the genes encoding for carbapenemases and ESBL individually in a 265

hypermucoviscous carbapenem-resistant K. pneumoniae strain KP_CRE23 using the 266

pCasKP/pSGKP system. The KP_CRE23 strain harbored one carbapenemase gene blaKPC-2 and 267

two ESBL genes blaSHV and blaCTX-M-65 (37). Because the KP_CRE23 strain is resistant to 268

kanamycin, the kanamycin marker in both the pSGKP-km and the pBECKP-km plasmids was 269

replaced with the spectinomycin marker, resulting in the pSGKP-spe and the pBECKP-spe 270

plasmids. As shown in Fig. 7A, we obtained the desired chromosomal blaSHV deletion mutant with 271

the efficiency of 4/12. However, in the case of the deletions of the plasmid-borne blaKPC-2 and 272

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blaCTX-M-65 genes using the same method, neither the desired gene-deletion bands nor the wild-type 273

bands were amplified by PCR in all the tested colonies (Fig. S10). The possible reason could be 274

that in the absence of a selection pressure, the DSB of the blaKPC-2-gene- and the 275

blaCTX-M-65-gene-carrying plasmids leaded to the plasmid removal without repair. It has been 276

reported that Cas9 nuclease-mediated DSB on plasmid can be used for plasmid removal in 277

Gram-negative bacteria (38). 278

Next, we attempted to use the base editor pBECKP-spe to inactivate the blaKPC-2 and 279

blaCTX-M-65 genes, because the pBECKP-spe system executed the editing by the introduction of a 280

single-stranded DNA break instead of a DSB. As shown in Fig. 7B-7C, the blaKPC-2 and blaCTX-M-65 281

genes were successfully mutated, resulting in the introduction of premature stop codons. The 282

editing efficiencies were 8/8 and 2/8, respectively. We then examined the imipenem (a major 283

carbapenem-class drug) susceptibility of the wild-type KP_CRE23 strain and three mutant strains 284

using the inhibition zone and the minimal inhibitory concentration (MIC) assays. As shown in Fig. 285

7D, the inactivation of the blaKPC-2 gene drastically increased the bacterial susceptibility to 286

imipenem, whereas no significant drug-susceptibility difference was observed when the blaSHV 287

gene was deleted or the blaCTX-M-65 gene was inactivated. These results verified that the blaKPC-2 288

gene was the key factor that contributed to the carbapenem resistance in the hypermucoviscous K. 289

pneumoniae strain KP_CRE23. These approaches can be applied to a more complex system to 290

dissect the drug-resistant mechanisms of K. pneumoniae. 291

292

DISCUSSION 293

Klebsiella pneumoniae is an important industrial microorganism and human pathogen, but 294

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the traditional genetic manipulation in K. pneumoniae is often time-consuming and laborious. 295

Therefore, more efficient and simple genetic tools are highly desirable. In this study, by harnessing 296

the powerful DNA cleavage ability of the engineered CRISPR-Cas9 system and the strong 297

recombination capacity of the lambda-Red system, we have developed a convenient and efficient 298

two-plasmid system pCasKP/pSGKP for iterative and scarless chromosomal gene deletion and 299

insertion in K. pneumoniae. We first constructed a single-plasmid CRISPR-Cas9 system, which 300

could efficiently cleave the genomic DNA of K. pneumoniae. Due to the lack of the 301

non-homologous end joining pathway, the DSB created by Cas9 nuclease on the chromosome was 302

lethal to K. pneumoniae. Although the repair templates had been supplied by cloning them into the 303

single CRISPR-Cas9 plasmid, no desirable deletion mutants were obtained, indicating the weak 304

capacity of the native homology-directed repair system of K. pneumoniae. To repair the DSB on 305

the chromosome created by the Cas9 nuclease, we introduced the lambda-Red recombination 306

system, which efficiently repaired the cleaved genomic DNA with the utilization of any type of 307

repair templates, including ssDNA. 308

To further simplify the editing process, we have developed a highly efficient cytidine base 309

editing system pBECKP by fusing a cytidine deaminase to the Cas9 nickase, enabling precise 310

C→T conversions in both the chromosome and the plasmids. The activity window and the 311

preference of the adjacent base of the editable sites were systematically investigated in K. 312

pneumoniae. The pBECKP system could irreversibly inactivate genes by converting four codons 313

(CAA, CAG, CGA and TGG) into premature stop codons in a programmable manner. One major 314

limitation of inactivating genes via the pBECKP system arises from the limited PAM sites that are 315

adjacent to the aforementioned four codons. The pBECKP requires the presence of a nearby NGG 316

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site located 13-18 bps away from an editable CAA, CAG, CGA or TGG codon. The recent 317

evolved xCas9 protein that recognizes a broad range of PAM sequences, including NG, GAA, and 318

GAT (39), may expand the base editing scope of the pBECKP system. 319

The pCasKP/pSGKP system and the pBECKP system have editing efficiencies in a variety of 320

clinically isolated K. pneumoniae strains. By using both the two editing systems, we verified the 321

carbapenem-resistant mechanism of a multidrug-resistant hypermucoviscous K. pneumoniae strain 322

KP_CRE23. Because the KP_CRE23 strain was resistant to kanamycin, the kanamycin marker in 323

both the pSGKP-km and the pBECKP-km plasmids was replaced with the spectinomycin marker. 324

Given that some K. pneumoniae isolates were insensitive to apramycin and/or kanamycin, the 325

antibiotic resistances may limit the applications of the pCasKP/pSGKP system and the pBECKP 326

system for genetic manipulations in multidrug-resistant K. pneumoniae strains. By testing the drug 327

sensitivity of several clinically isolated K. pneumoniae strains, we detected that the majority of 328

them were sensitive to hygromycin B. Thereby we constructed a new plasmid pCasKP-hph, which 329

could serve as an alternative option for genetic manipulation in those apramycin-resistant K. 330

pneumoniae strains. The antibiotic marker could also be replaced to any other suitable selection 331

marker for the editing in different K. pneumoniae strains. 332

The off-target effect was rarely noticed for DSB-based genome editing in NHEJ-deficient 333

bacteria, because the cells with off-target events can not survive. However, the base editor directly 334

mutated the target site without generating a DSB, potential off-target effects were not lethal to the 335

cells edited by pBECKP system. To obtain a high editing efficiency and reduce potential off-target 336

effects, the spacers used in this study were designed using the sgRNAcas9 software (40). The 337

sgRNAcas9 software could screen all the suitable spacer sequences in the target genes and 338

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evaluate their potential off-target sites throughout the entire K. pneumoniae genome. 339

Overall, we have engineered the two-plasmid system pCasKP/pSGKP for genome editing 340

and the single-plasmid system pBECKP for base editing in a variety of K. pneumoniae strains. 341

Given the simple construction procedures and high efficiency, future applications of the two 342

editing systems should dramatically facilitate a wide variety of investigations in K. pneumoniae 343

and relevant enterobacteriaceae species, such as gene characterization, drug discovery, and 344

metabolic engineering. 345

346

MATERIALS AND METHODS 347

Plasmids, bacterial strains, primers, and growth conditions. All of the plasmids used in 348

this study are listed in Table 1. All of the bacterial strains used in this study are listed in Table 2. 349

The primers used in this study were purchased from GENEWIZ (Suzhou, China) and are listed in 350

Table S1. E. coli DH5α and K. pneumoniae strains were grown in lysogeny broth (LB) medium 351

(per liter, 5 g of yeast extract, 10 g of tryptone, 10 g of NaCl, pH 7.2~7.4). Antibiotics were added 352

at the following concentrations: apramycin 30-50 µg/mL, hygromycin B 100 µg/mL, kanamycin 353

50 µg/mL, and spectinomycin 50-100 µg/mL for both E. coli and K. pneumoniae strains. 354

Plasmid construction. The temperature-sensitive pCasKP-apr plasmid was constructed using 355

the following procedures. The rpsL promoter was PCR-amplified from the genomic DNA of the K. 356

pneumoniae strain KP_1.6366. The gene encoding the Cas9 nuclease was amplified from the 357

pCasSA plasmid (41). The aforementioned two fragments along with the NdeI_linearized 358

pKOBEG-apr plasmid (23) were assembled together using In-fusion Cloning, resulting in the final 359

plasmid pCasKP-apr plasmid. The pCasKP-hph plasmid was constructed by replacing the 360

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apramycin-resistance gene of the pCasKP-apr plasmid with the hygromycin B-resistance gene. 361

The sgRNA-expression cassette was synthesized commercially by GENEWIZ (Suzhou, China). 362

The cassette contained three elements: the constitutive J23119 promoter, two BsaI restriction sites 363

for the insertion of 20 bp spacer, and the sgRNA scaffold. The sgRNA-expression cassette was 364

cloned into the EcoRV-digested pUC57 vector, yielding the pUC57-sgRNA plasmid. Then the 365

sacB gene amplified from the pCasPA plasmid (42) was inserted into the HindIII-digested 366

pUC57-sgRNA plasmid via In-fuison Cloning, resulting in the final PSGKP-km plasmid. The 367

pSGKP-spe plasmid was constructed by replacing the kanamycin-resistance gene of the 368

pSGKP-km plasmid with spectinomycin-resistance gene. 369

The pBECKP-km plasmid was constructed with the following procedure. The low-copy plasmid 370

backbone containing pBR322_origin, the rop gene and the kanamycin-resistance marker were 371

amplified from the pET28a plasmid. The sgRNA-expression cassette and the sacB gene were 372

amplified from the pSGKP-km plasmid. The two fragments were assembled into a plasmid by 373

In-fuison Cloning. Finally, the BEC-nCas9 cassette amplified from pnCasSA-BEC plasmid (32) 374

was inserted into the HindIII site of the aforementioned plasmid to form the final all-in-one 375

pBECKP-km plasmid. The pBECKP-spe plasmid was constructed by replacing the 376

kanamycin-resistance gene of the pBECKP-km plasmid with the spectinomycin-resistance gene. 377

All the plasmids constructed in this study were validated by PCR, enzyme digestion, and 378

DNA sequencing. Their sequences were submitted to GenBank database under the accession 379

numbers MH587683 (pSGKP-km), MH587684 (pSGKP-spe), MH587685 (pBECKP-km), 380

MH587686 (pBECKP-spe), MH587687 (pCasKP-apr), and MH587688 (pCasKP-hph). All the 381

plasmids constructed in this study will be available in Addgene with the numbers of 117231 382

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(pCasKP-apr), 117232 (pCasKP-hph), 117233 (pSGKP-km), 117234 (pSGKP-spe), 117235 383

(pBECKP-km), and 117236 (pBECKP-spe), respectively. 384

Preparation of competent Cells and electroporation. For the K. pneumoniae wild-type 385

strain, 1 mL overnight culture from a fresh single colony was diluted into 100 mL of LB broth and 386

incubated at 37 °C. When the optical density at 600 nm (OD600) of the cell culture reached at 0.5 387

to 0.7, the culture was immediately chilled on ice for 20 min and then harvested by centrifugation 388

at 7200 g for 5 min. The supernatant was discarded, and the cells were resuspended by pipetting 389

gently with 15 mL of sterile ice-cold 10% glycerol. The centrifugation and resuspension steps 390

were repeated twice. Finally, the cells were resuspended with 1 mL of ice-cold 10% glycerol. 50 391

μL aliquots were frozen in liquid nitrogen and stored at −80 °C. 392

For the pCasKP-harboring K. pneumoniae strain, 1 mL overnight culture from a fresh single 393

colony was diluted into 100 mL of LB broth containing 30 µg/mL apramycin and incubated at 394

30 °C. When the cell density reached an OD600 of approximately 0.2, 1 mL of 20% L-arabinose 395

was added for induction of the lambda-Red recombineering operon of pCasKP. After induction at 396

30 °C for 2 hours, the culture was prepared as electrocompetent cells in a similar way as that of 397

the wild-type K. pneumoniae. 398

For electroporation, 50 µL of electrocompetent cells were thawed on ice for several minutes. 399

Then the cells were mixed with no more than 5 µL plasmid or donor template. The mixture was 400

transferred into a 2 mm electroporation cuvette (Bio-Rad) and electroporated at 2.5 kV, 200 Ω, and 401

25 µF. After being pulsed, the cells were recovered in 1 mL antibiotic-free LB broth and incubated 402

at 30 °C for 1.5 h before being plated onto LB agar plates supplemented with the required 403

antibiotics. The plates were incubated at 30 °C overnight. 404

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Genome editing and base editing. The detailed protocols for spacer-cloning, genome 405

editing, base editing and plasmids curing in K. pneumoniae were provided in the supplementary 406

material. 407

Antimicrobial susceptibility assay. For the inhibition testing, fresh K. pneumoniae 408

suspension was adjusted to 0.5 mihms turbidity and then diluted 10 times with saline. The diluted 409

bacterial suspension was evenly coated onto a MH agar plate. The plate was dried for 5 min. A 410

fosfomycin (50 µg/tablet, OXOID) or an imipenem (10 µg/tablet, OXOID) tablet was placed in 411

the center of the aforementioned MH plate. The plate was incubated at 35 °C for 20 h to produce 412

the inhibition zones. 413

For the minimal inhibitory concentration (MIC) assay, the MICs of imipenem for the 414

carbapenem-resistant KP_CRE23 strain and three mutant strains were determined using the 415

96-well broth microdilution method recommended by the Clinical and Laboratory Standards 416

Institute. In brief, fresh K. pneumoniae suspension was adjusted to 0.5 mihms turbidity and then 417

diluted 10 times with saline. The 2 µL diluted solutions with 5.0×106 CFU bacterial cells were 418

inoculated into 200 µL MH liquid medium containing serial twofold dilution concentrations of the 419

imipenem (0.5 ~ 64 µg/mL). The imipenem-free MH liquid medium was used as the control. After 420

incubation at 35 °C for 20 h, the MICs of complete growth inhibition were determined by visual 421

inspection. 422

423

SUPPLEMENTAL MATERIAL 424

Supplemental material for this article may be found at xxx. 425

426

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ACKNOWLEDGMENTS 427

We thank Dr. Jian Hao from Shanghai Advanced Research Institute, Chinese Academy of 428

Sciences, for generously providing the K. pneumoniae KP_1.6366 strain. 429

This work was financially supported by the National Key R&D Program of China 430

(2017YFA0506800), the National Natural Science Foundation of China (91753127, 31700123), 431

the Shanghai Comittee of Science and Technology, China (17ZR1449200), the ShanghaiTech 432

Startup Funding, and the “Young 1000 Talents” Program (to Q.J.); the China Postdoctoral Science 433

Foundation (2018M632190) (to Y.W.), and the Shanghai Sailing Program (18YF1416500) (to 434

W.C.). 435

436

DECLARATION OF INTERESTS 437

Two patent applications have been submitted for the two-plasmid genome editing system 438

pCasKP/pSGKP and the single-plasmid base editing system pBECKP. 439

440

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Table 1 Plasmids used in this study. 558

Plasmids Description Reference

pCasSA Plasmid carries the bacterial Cas9 nuclease gene,

Kmr, Cm

r

(41)

pKOBEG-apr Thermosensitive plasmid, carries lambda-Red

genes, Aprr

(23)

pOSCAR Cloning plasmid, Sper (43)

pCasPA Plasmid carries the sacB gene, Tetr (42)

pnCasSA-BEC Plasmid carries the bec-Cas9D10A gene, Kmr, Cm

r (32)

pET28a Low-copy plasmid, Kmr Lab stock

pMD19-mcherry Plasmid carries the mcherry gene, Ampr Lab stock

pMD19-hyg Plasmid carries the hph gene, Ampr, Hyg

r Lab stock

pCas-sgRNAKP K. pneumoniae single-plasmid CRISPR-Cas9

editing vector, Aprr

This study

pCas-sgRNAKP_dhaF pCas-sgRNAKP derivative with dhaF spacer This study

pCas-sgRNAKP_dhaF_HR pCas-sgRNAKP derivative with dhaF spacer and ~

1 kb each repair arms

This study

pCasKP-apr Thermosensitive plasmid, expresses Cas9 and

lambda-Red proteins in K. pneumoniae, Aprr

This study

pCasKP-hph Thermosensitive plasmid, expresses Cas9 and

lambda-Red proteins in K. pneumoniae, Hygr

This study

pSGKP-km Plasmid express sgRNA in K. pneumoniae, Kmr This study

pSGKP-spe Plasmid express sgRNA in K. pneumoniae, Sper This study

pSGKP_dhaF pSGKP-km derivative with dhaF spacer This study

pSGKP_dhaF_HR pSGKP-km derivative with dhaF spacer and ~ 1 kb

each repair arms

This study

pSGKP_dhaK pSGKP-km derivative with dhaK spacer This study

pSGKP_dhaK_HR pSGKP-km derivative with dhaF spacer and ~ 0.5

kb each repair arms

This study

pSGKP_fosA pSGKP-km derivative with fosA spacer This study

pSGKP_pyrF pSGKP-km derivative with pyrF spacer This study

pSGKP_fepB pSGKP-km derivative with fepB spacer This study

pSGKP_ramA pSGKP-km derivative with ramA spacer This study

pSGKP-spe_blaKPC pSGKP-spe derivative with blaKPC spacer This study

pSGKP-spe_blaSHV pSGKP-spe derivative with blaSHV spacer This study

pSGKP-spe_blaCTX pSGKP-spe derivative with blaCTX spacer This study

pBECKP-km K. pneumoniae base editing vector, Kmr This study

pBECKP-spe K. pneumoniae base editing vector, Sper This study

pBECKP_fosA_1 pBECKP-km derivative with fosA spacer 1 This study

pBECKP_fosA_2 pBECKP-km derivative with fosA spacer 2 This study

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pBECKP_fosA_3 pBECKP-km derivative with fosA spacer 3 This study

pBECKP_dhaK_1 pBECKP-km derivative with dhaK spacer 1 This study

pBECKP_dhaK_2 pBECKP-km derivative with dhaK spacer 2 This study

pBECKP_dhaK_3 pBECKP-km derivative with dhaK spacer 3 This study

pBECKP_dhaK_4 pBECKP-km derivative with dhaK spacer 4 This study

pBECKP_dhaF_1 pBECKP-km derivative with dhaF spacer 1 This study

pBECKP_dhaF_2 pBECKP-km derivative with dhaF spacer 2 This study

pBECKP_dhaF_3 pBECKP-km derivative with dhaF spacer 3 This study

pBECKP_dhaF_4 pBECKP-km derivative with dhaF spacer 4 This study

pBECKP_dhaF_5 pBECKP-km derivative with dhaF spacer 5 This study

pBECKP-spe_blaSHV pBECKP-spe derivative with blaSHV spacer This study

pBECKP-spe_blaCTX pBECKP-spe derivative with blaCTX spacer This study

559

560

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Table 2 Bacterial strains used in this study. 561

Strains Description Reference

E. coli DH5α F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1

endA1 hsdR17 (rK–, mK+) phoA supE44 lambda–

thi-1 gyrA96 relA1

Lab stock

KP_1.6366 a wild-type industrial strain of K. pneumoniae (44)

KP_1.6366dhaF KP_1.6366 ΔdhaF This study

KP_1.6366dhaK KP_1.6366 ΔdhaK This study

KP_1.6366fosA KP_1.6366 ΔfosA This study

KP_1.6366fosA::mcherry KP_1.6366 ΔfosA::mcherry This study

KP_1.6366dhaFdhaK KP_1.6366 ΔdhaFΔdhaK This study

KP_1.6366fosA W92 to

stop

KP_1.6366 fosA W92 mutation to stop codon This study

KP_3744 a wild-type clinically isolated strain of K.

pneumoniae

Lab stock

KP_3744pyrF KP_3744ΔpyrF This study

KP_3744fepB KP_3744ΔfepB This study

KP_3744ramA KP_3744ΔramA This study

KP_5573 a wild-type clinically isolated strain of K.

pneumoniae

Lab stock

KP_5573fosA KP_5573 ΔfosA This study

KP_5573pyrF KP_5573 ΔpyrF This study

KP_5573fepB KP_5573 ΔfepB This study

KP_5573ramA KP_5573 ΔramA This study

KP_5573fosA W92 to stop KP_5573 fosA W92 mutation to stop codon This study

KP_CRE23 a wild-type clinically isolated strain of K.

pneumoniae with multidrug-resistance and

hypermucoviscosity

(37)

KP_CRE23blaSHV KP_CRE23 ΔblaSHV This study

KP_CRE23blaKPC-2 W164

to stop

KP_CRE23 blaKPC-2 W164 mutation to stop codon This study

KP_CRE23blaCTX-M-65

Q136 to stop

KP_CRE23 blaCTX-M-65 Q136 mutation to stop codon This study

562

563

564

565

566

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567

FIG 1 The CRISPR-Cas9 system is functional in K. pneumoniae. The dhaF-pacer-introduced 568

pCas9-sgRNAKP plasmid (pCas9-sgRNAKP_dhaF) efficiently killed the K. pneumoniae cells 569

(middle). Genome editing using both the dhaF-spacer-and the repair arm-introduced 570

pCas-sgRNAKP plasmid (pCas9-sgRNAKP_dhaF_HR) did not yield the desired recombinants 571

(right). An empty pCas9-sgRNAKP plasmid was transformed into the KP_1.6366 strain as a 572

control (left). 573

574

575

576

577

578

579

580

581

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582

FIG 2 Genome editing in K. pneumoniae using a two-plasmid pCasKP/pSGKP system. (A) 583

Scheme for the CRISPR-Cas9 and lambda-Red recombination-mediated genome editing method. 584

The sgRNA-Cas9 complex cleaves the double-strand DNA proximal to a PAM site, generating a 585

double-stranded DNA break. The double-stranded DNA break is repaired via 586

lambda-Red-mediated homologous recombination using a donor template. (B) Maps of the 587

pCasKP-apr and pSGKP-km plasmids. pCasKP-apr contains the Cas9 gene with a constitutive 588

rpsL promoter, the lambda-Red recombination genes (gam, bet and exo) with an L-arabinose 589

inducible promoter ParaB and the temperature-sensitive replication repA101ts. pSGKP-km 590

contains the sgRNA with the synthetic J23119 promoter and the sacB gene for plasmid curing. (C) 591

The two-plasmid system pCasKP/pSGKP enabled highly efficient gene deletion in the industrial K. 592

pneumoniae strain KP_1.6366. The deletion efficiency of the dhaF gene was 20/20. (D) The CFUs 593

of each transformation using different types of donor templates in the KP_1.6366 strain. 200 ng 594

dhaK-spacer-introduced pSGKP_dhaK plasmid, 300 ng pSGKP_dhaK_HR plasmid containing the 595

repair template (~500 bp each), 200 ng pSGKP_dhaK plasmid with 300 ng dsDNA repair template 596

(~500 bp each), and 200 ng pSGKP_dhaK plasmid with 300 μM ssDNA (90 nt) were used for the 597

transformations, respectively. Error bars represent standard deviation among three independent 598

experiments. 599

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600

FIG 3 Scheme of the procedures for the iterative editing of the pCasKP/pSGKP system. For new 601

rounds of genome editing, the spacer-introduced pSGKP-km plasmid can be recycled by 602

cultivation in the presence of sucrose. After all the desired editing, both the plasmids can be cured 603

by culturing the cells at 37 °C and in the presence of sucrose. Apr: apramycin; Km: kanamycin. 604

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605

FIG 4 The two-plasmid pCasKP/pSGKP system allowed for highly efficient genome editing in 606

the clinically isolated K. pneumoniae strains. (A) The deletion of the pyrF gene in the KP_3744 607

strain. The editing efficiency was 10/10. The lane of CK was the PCR band from the wild-type 608

strain. The growth defect on the synthetic CDM plate containing no uracil indicated the disruption 609

of the pyrF gene. (B) The deletion of the fepB gene in the KP_3744 strain. The editing efficiency 610

was 10/10. (C) The deletion of the fosA gene in the KP_5573 strain. The editing efficiency was 611

10/10. The deletion of the fosA gene was confirmed by both the PCR and the tablet diffusion assay. 612

(D) The deletion of the ramA gene in the KP_5573 strain. The editing efficiency was 10/10. 613

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614 FIG 5 The pBECKP system enabled highly efficient base editing in K. pneumoniae. (A) Scheme 615

of the procedures of pBECKP-mediated base editing. The Cas9 nickase cleaves the non-edited 616

strand and the APOBEC1 deaminase catalyzes the conversion of C to U. The resulting U:G 617

heteroduplex can be permanently converted to the T:A base pair by DNA repair or replication. (B) 618

Map of the pBECKP-km plasmid. The pBECKP-km plasmid contains the 619

rAPOBEC1-XTEN-Cas9D10A fusion gene, the sgRNA expression cassette, the sacB gene, and the 620

copy-number-limiting gene rop. (C) W92 of the fosA gene in the KP_5573 strain was successfully 621

mutated to a stop codon with the efficiency of 8/8 using the pBECKP system. A representative 622

sequencing chromatogram for the fosA mutant was shown. The similar fosfomycin-inhibition-zone 623

diameters between the deletion-mutant strain and two point-mutant strains indicated the successful 624

disruption of the fosA gene. (D) Alignments of the editing products of a C-rich locus by the 625

pBECKP system. The mutated Ts are colored red. The Cs at different positions were mutated to Ts 626

with different efficiencies by the pBECKP system. 627

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628

FIG 6 Determination of the activity window and sequence context preference of the pBECKP 629

system in K. pneumoniae. The Cs with high editing efficiencies were marked with red squares. A 630

few Cs at position 9 of the spacer8 and position 7 of the spacer10 were mutated to As, but not Ts. 631

These sites were colored green. 632

633

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FIG 7 blaKPC-2 is the key factor for carbapenem resistance of a multidrug-resistant 635

hypermucoviscous K. pneumoniae strain KP_CRE23. (A) pCasKP/pSGKP-mediated deletion of 636

the chromosomal blaSHV gene. The deletion efficiency was 4/12. (B-C) W164 of the plasmid-borne 637

blaKPC-2 gene (B) and Q136 of the plasmid-borne blaCTX-M-65 gene (C) were successfully mutated 638

to stop codons with efficiencies of 8/8 and 2/8, respectively, by the pBECKP system. (D) blaKPC-2 639

was the key gene for carbapenem resistance of the K. pneumoniae strain KP_CRE23. 640

641

642

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