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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
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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
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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
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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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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
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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
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