CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase EnablePrecise and Efficient Genome Editing in Klebsiella pneumoniae

Yu Wang,a Shanshan Wang,b Weizhong Chen,a Liqiang Song,a Yifei Zhang,a Zhen Shen,c Fangyou Yu,d Min Li,c

Quanjiang Jia

aSchool of Physical Science and Technology, ShanghaiTech University, Shanghai, ChinabDepartment of Laboratory Medicine, Wenzhou Medical University, Wenzhou, ChinacDepartment of Laboratory Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University,Shanghai, China

dDepartment of Clinical Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University,Shanghai, China

ABSTRACT Klebsiella pneumoniae is a promising industrial microorganism as wellas a major human pathogen. The recent emergence of carbapenem-resistant K.pneumoniae has posed a serious threat to public health worldwide, emphasizinga dire need for novel therapeutic means against drug-resistant K. pneumoniae.Despite the critical importance of genetics in bioengineering, physiology studies,and therapeutic-means development, genome editing, in particular, the highly desir-able scarless genetic manipulation in K. pneumoniae, is often time-consuming andlaborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for preciseand iterative genome editing in K. pneumoniae. By harnessing the clustered regularlyinterspaced short palindromic repeat (CRISPR)-Cas9 genome cleavage system andthe lambda Red recombination system, pCasKP-pSGKP enabled highly efficient ge-nome editing in K. pneumoniae using a short repair template. Moreover, we devel-oped a cytidine base-editing system, pBECKP, for precise C¡T conversion in boththe chromosomal and plasmid-borne genes by engineering the fusion of the cyt-idine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP-pSGKPand the pBECKP tools, the blaKPC-2 gene was confirmed to be the major factorthat contributed to the carbapenem resistance of a hypermucoviscouscarbapenem-resistant K. pneumoniae strain. The development of the two editingtools will significantly facilitate the genetic engineering of K. pneumoniae.

IMPORTANCE Genetics is a key means to study bacterial physiology. However,the highly desirable scarless genetic manipulation is often time-consumingand laborious for the major human pathogen K. pneumoniae. We developed aCRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system,enabling rapid, highly efficient, and iterative genome editing in both industrial andclinically isolated K. pneumoniae strains. We applied both tools in dissecting thedrug resistance mechanism of a hypermucoviscous carbapenem-resistant K. pneu-moniae strain, elucidating that the blaKPC-2 gene was the major factor that con-tributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant K. pneumoniae strain. Utilization of the two tools will dramaticallyaccelerate a wide variety of investigations in diverse K. pneumoniae strains andrelevant Enterobacteriaceae species, such as gene characterization, drug discov-ery, and metabolic engineering.

KEYWORDS CRISPR, Cas9, Klebsiella pneumoniae, genetic engineering, genomeediting, base editing

Received 26 July 2018 Accepted 10September 2018

Accepted manuscript posted online 14September 2018

Citation Wang Y, Wang S, Chen W, Song L,Zhang Y, Shen Z, Yu F, Li M, Ji Q. 2018. CRISPR-Cas9 and CRISPR-assisted cytidine deaminaseenable precise and efficient genome editing inKlebsiella pneumoniae. Appl Environ Microbiol84:e01834-18. https://doi.org/10.1128/AEM.01834-18.

Editor Harold L. Drake, University of Bayreuth

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Quanjiang Ji,quanjiangji@shanghaitech.edu.cn.

METHODS

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Klebsiella pneumoniae is a high-GC-content Gram-negative bacillus of the Enterobac-teriaceae family and is widely distributed in the natural environment and on the

mucosal surfaces of mammals. It is considered a promising industrial microorganismbecause of its capacity to naturally synthesize a diverse range of valuable chemicals (1).In addition, K. pneumoniae is a major human pathogen, causing a wide variety ofhospital- and community-acquired infections, such as pneumonia, bacteremia, andurinary tract infections (2). In recent years, the emergence of hypermucoviscous andmultidrug-resistant K. pneumoniae strains, in particular, carbapenem-resistant hyper-mucoviscous K. pneumoniae strains, has posed a severe public health crisis worldwide(3–5). Thereby, novel therapeutic means against multidrug-resistant K. pneumoniaeinfections are urgently needed.

The development of novel therapeutic means against drug-resistant K. pneumoniaeinfections would benefit greatly from efficient and convenient genome editing andscreening tools, which allow effective identification of the key genes and pathwaysresponsible for bacterial virulence and drug resistance. Although the lambda Redrecombination system has been developed and widely utilized for genetic manipula-tion and the clustered regularly interspaced short palindromic repeat (CRISPR) inter-ference (CRISPRi) system has been developed recently for transcriptional inhibition in K.pneumoniae, the highly desirable scarless and precise genome editing in K. pneumoniaeis still time-consuming and laborious (6, 7). For instance, to construct a markerlessdeletion mutant in K. pneumoniae, a target gene is first replaced by an antibiotic markervia a double-crossover homologous recombination process mediated by the lambdaRed recombination proteins (Gam, Bet, and Exo). Second, the antibiotic marker iseliminated by the utilization of a helper plasmid expressing the FLP recombinase (FRT).The FLP recombinase directly binds to the repeated FLP recognition sites flanking theantibiotic gene and catalyzes the elimination reaction, leaving an FRT scar in the placeof the target gene. When multiple rounds of genetic modification are performed usingthe aforementioned method, the introduction of multiple FRT scars in the genome maylead to genome instability by causing genome rearrangement (8).

The recently discovered CRISPR-Cas9 system allows for the efficient generation of adouble-strand break (DSB) at a desired site of the target genome (9, 10), thereby raisingthe possibility of one-step scarless genome editing in K. pneumoniae. The CRISPRsystem is an adaptive immune system and is utilized by bacteria and archaea to fightagainst invading phages and foreign plasmids (11, 12). The wildly utilized CRISPR-Cas9system is composed of two components, the Cas9 nuclease from Streptococcus pyo-genes and a single artificial chimeric guide RNA (sgRNA) (13). The sgRNA directs theCas9 protein to a target genomic locus through complementary base pairing to a targetsequence in the presence of a downstream 5=-NGG-3= protospacer adjacent motif(PAM) (14). After that, the Cas9 nuclease creates a DSB within the base pairing region(13). Given the lack of the nonhomologous end-joining (NHEJ) pathway in mostbacteria, including Klebsiella pneumoniae, chromosomal cleavage is lethal to bacterialcells unless it is repaired by the homologous recombination (HR) pathway with theutilization of exogenously supplied donor DNA repair templates (15). Thereby, precisegenetic manipulation, including gene deletions, point mutations, and gene insertions,can be achieved by simply customizing an approximately 20-nucleotide (nt) spacersequence and a designed donor repair template.

Furthermore, the recently developed CRISPR RNA-guided deaminase systems enableprecise base editing, opening a new avenue for genome editing in biology. Until now,two kinds of base editors have been developed, the cytidine editor BEC (16, 17) and theadenosine editor ABE (18). Each base editor is composed of a Cas9 nickase (D10A in thecase of S. pyogenes Cas9 [SpCas9]) or a dead Cas9 protein (D10A and H840A in the caseof SpCas9) and a deaminase fused to the Cas9 protein. Relying on the base pairingbetween a target sequence and the 20-nt guide RNA sequence, the tethered deaminasecan be directed to any target locus to perform nucleoside deamination through adeamination reaction (C¡U for the BEC editor and A¡I for the ABE editor). In livingcells, the DNA repair or replication mechanism would efficiently convert the U:G or I:T

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heteroduplex pair to the desired T·A or G·C pair. Distinct from the CRISPR-Cas9-mediated genome editing, the base editors directly catalyze the conversions of nucleo-sides without the formation of DSB or the utilization of a donor template.

In this study, we developed a CRISPR-Cas9-mediated genome-editing method anda base-editing system enabling rapid, highly efficient, and iterative genome editing inboth industrial and clinically isolated K. pneumoniae strains. By using both genome-editing tools, we confirmed that the blaKPC-2 gene was the major factor that contributedto the carbapenem resistance of a hypermucoviscous carbapenem-resistant K. pneu-moniae strain. The development of these genome-editing tools will dramatically accel-erate a wide variety of investigations in K. pneumoniae.

RESULTSEstablishment of a single-plasmid CRISPR-Cas9 system in K. pneumoniae. To

develop a convenient and scarless genetic manipulation method in K. pneumoniae, wefirst sought to harness the CRISPR-Cas9 system for genome editing. To access thefunctionality of the CRISPR-Cas9 system in K. pneumoniae, we constructed a single-plasmid system, pCas9-sgRNAKP, that expressed both the well-studied Streptococcuspyogenes Cas9 protein and the sgRNA in the same plasmid (19). The transformation ofthe empty pCas9_sgRNAKP plasmid into K. pneumoniae yielded a lawn of colonies,whereas the transformation of the nonessential dhaF spacer-introduced pCas9-sgRNAKP plasmid only produced a few colonies (Fig. 1), strongly indicating the effectivecleavage of bacterial genome by the CRISPR-Cas9 system.

Next, we assembled the repair templates (�1 kb each) of the dhaF gene into thedhaF spacer-introduced pCas9-sgRNAKP plasmid to test the functionality of the systemfor gene deletion in K. pneumoniae. The transformation of the assembled plasmid intoK. pneumoniae yielded only fewer than 5 colonies (Fig. 1). Further PCR screeninganalysis revealed that none of them were the desired deletion mutants, indicating thatthe intrinsic homologous recombination capacity of K. pneumoniae was not greatenough for the direct repair of the lethiferous double-stranded DNA break of thegenome.

To alleviate the toxicity of chromosomal cleavage by the Cas9 nuclease,two versions of Cas9 nickase expression plasmids, pnCas9D10A_sgRNAKP andpnCas9H840A_sgRNAKP, were constructed by mutating the active sites of Cas9protein Aps10 or His840 to Ala, respectively. The transformations of the Cas9nickase plasmids containing both the dhaF spacer and the corresponding repairtemplate (�1 kb each) indeed yielded plenty of colonies. However, PCR screening andfurther sequencing revealed that no desired homologous recombination-repair eventswere observed (see Fig. S1 in the supplemental material). It is likely that K. pneumoniaepreferred to accurately repair the DNA nick using the complementary strand, ratherthan the exogenous donor templates.

Development of the two-plasmid system pCasKP-pSGKP for genome editing.Phage recombination systems, such as lambda Red and Rac-RecET, possess a strongerrecombination capacity than that of normal bacterial cells (20, 21). We sought to

FIG 1 The CRISPR-Cas9 system is functional in K. pneumoniae. The dhaF spacer-introduced pCas9-sgRNAKP plasmid (pCas9-sgRNAKP_dhaF) efficiently killed the K. pneumoniae cells (middle). Genomeediting using both the dhaF spacer- and the repair arm-introduced pCas-sgRNAKP plasmid (pCas9-sgRNAKP_dhaF_HR) did not yield the desired recombinants (right). An empty pCas9-sgRNAKP plasmidwas transformed into the KP_1.6366 strain as a control (left).

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increase the homologous recombination capacity of K. pneumoniae by introducing thephage lambda Red recombination system into the bacteria. To achieve this, wedesigned and constructed a two-plasmid system, pCasKP-pSGKP (Fig. 2A and B). ThepCasKP plasmid expressed the Cas9 protein under the control of the constitutive K.pneumoniae rpsL promoter and the lambda Red recombination proteins (Gam, Bet, andExo) under the control of an L-arabinose-inducible ParaB promoter. The pSGKP plasmidexpressed the sgRNA under the control of the synthetic constitutive J23119 promoter(22). Two reversed BsaI sites were inserted between the J23119 promoter and thesgRNA scaffold for the seamless and one-step assembly of spacers (Fig. S2). In addition,

FIG 2 Genome editing in K. pneumoniae using a two-plasmid pCasKP-pSGKP system. (A) Scheme for the CRISPR-Cas9 and lambda Red recombination-mediatedgenome-editing method. The sgRNA-Cas9 complex cleaves the double-strand DNA proximal to a PAM site, generating a double-stranded DNA break. Thedouble-stranded DNA break is repaired via lambda Red-mediated homologous recombination using a donor template. (B) Maps of the pCasKP-apr andpSGKP-km plasmids. pCasKP-apr contains the Cas9 gene with a constitutive rpsL promoter, the lambda Red recombination genes (gam, bet, and exo) with anL-arabinose-inducible promoter ParaB, and the temperature-sensitive replicon repA101(Ts) (repA101ts). pSGKP-km contains the sgRNA with the synthetic J23119promoter and the sacB gene for plasmid curing. (C) The two-plasmid system pCasKP-pSGKP enabled highly efficient gene deletion in the industrial K.pneumoniae strain KP_1.6366. The deletion efficiency of the dhaF gene was 20/20. (D) The CFU of each transformation using different types of donor templatesin the KP_1.6366 strain. Two hundred nanograms of dhaK spacer-introduced pSGKP_dhaK plasmid, 300 ng pSGKP_dhaK_HR plasmid containing the repairtemplate (�500 bp each), 200 ng pSGKP_dhaK plasmid with 300 ng dsDNA repair template (�500 bp each), and 200 ng pSGKP_dhaK plasmid with 300 �MssDNA (90 nt) were used for the transformations shown from left to right, respectively. Error bars represent standard deviation from the results from threeindependent experiments.

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the temperature-sensitive replicon repA101(Ts) (23) and the sucrose-sensitive gene sacB(24) were introduced into the pCasKP and pSGKP plasmids, respectively, for easyplasmid curing after editing.

To assess the genome-editing ability of the constructed two-plasmid system, wesought to delete the dhaF gene in the industrial K. pneumoniae strain KP_1.6366. To dothis, the pCasKP-apr plasmid was first electroporated into the wild-type industrial K.pneumoniae strain KP_1.6366 to obtain the pCasKP-apr-harboring strain. After theinduction of L-arabinose, the cells containing the pCasKP-apr plasmid were collectedand prepared as the competent cells. Then, the dhaF deletion plasmid pSGKP-dhaF-HRwas transformed into the aforementioned pCasKP-apr-harboring competent cells byelectroporation. The pSGKP-dhaF-HR plasmid was constructed by assembling both thedhaF spacer and the repair arms of the dhaF gene (�1 kb each) into the pSGKP-kmplasmid. The subsequent transformation yielded �1,000 colonies. Twenty colonieswere randomly picked to test the editing efficiency. As shown in Fig. 2C, successfuldeletion of the dhaF gene was confirmed in all the picked colonies by both PCR andsequencing.

To simplify the plasmid construction procedures and accelerate the genome-editingprocess, we attempted to utilize the linear homologous DNA fragment as the repairtemplate. The lambda Red recombination system is capable of using the plasmid-bornedonor DNA, linear double-stranded DNA (dsDNA), or single-stranded DNA (ssDNA) asthe repair template (25). To evaluate the editing efficiency of linear repair templates, alinear dsDNA (�500 bp each) and an ssDNA (45 nt each) were cotransformed individ-ually with the dhaK spacer-introduced pSGKP-km plasmid (pSGKP_dhaK) into theL-arabinose-induced pCasKP-apr-harboring cells to delete the dhaK gene (26). The trans-formations of the pSGKP_dhaK plasmid (only dhaK spacer) and the pSGKP_dhaK_HRplasmid (dhaK spacer and assembled with �500 bp each repair template) were used as thenegative and positive controls, respectively. As shown in Fig. 2D and S3A, �1,000colonies were observed for all the transformations containing any type of the donorrepair templates, whereas fewer than 10 colonies were obtained for the transformationwithout a repair template. Further PCR screening and sequencing showed that thedeletion efficiencies were 100% for all the transformations containing the repairtemplates (Fig. S3B to D). Moreover, we assessed the capacity of the two-plasmidsystem pCasKP-pSGKP to delete the fosA gene with the utilization of ssDNA as therepair template (27). As shown in Fig. S4, the editing efficiency was 9/10.

In addition to gene deletion, the two-plasmid system pCasKP-pSGKP was used forgene insertion in K. pneumoniae. We attempted to replace the fosA gene with themcherry gene. We cotransformed the fosA spacer-introduced pSGKP-km plasmid(pSGKP-fosA) and the mcherry gene with 45-bp homology extensions into the pCasKP-apr-harboring KP_1.6366 strain by electroporation (Fig. S5A). More than 100 colonieswere recovered, and the insertion efficiency was 9/10 (Fig. S5B). Together, theseexperiments demonstrated that the two-plasmid pCasKP-pSGKP system possessed agreat capacity for genetic manipulation in K. pneumoniae with the utilization of a shortrepair template.

Complicated physiology study and metabolic engineering of K. pneumoniae requirethe genetic manipulation of multiple genes, thereby requiring multiple rounds ofgenome editing. For the second-round editing, the spacer-incorporated pSGKP-kmplasmid needs to be recycled for different target loci, while the pCasKP-apr plasmid canbe maintained to express the Cas9 protein and the lambda Red recombination system(Fig. 3). We inoculated one colony containing the desired dhaK deletion into lysogenybroth (LB) medium with the supplementation of apramycin. The cells were cultured at30°C overnight. Next, a fraction of the cells was streaked onto an LB agar platecontaining apramycin and sucrose and incubated at 30°C until colonies were visible. Asshown in Fig. S6A, all the four randomly picked colonies could only grow normally onthe plate containing apramycin, whereas none of them could grow on the platecontaining both apramycin and kanamycin, confirming the successful removal of thepSGKP-km plasmid with the maintenance of the pCasKP-apr plasmid. Next, the dhaF

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gene was deleted in the pSGKP-dhaK-cured cells with an efficiency of 10/10 (Fig. S6B).After finishing all the desired genome editing, both the pCasKP-apr and the pSGKP-kmplasmids could be easily cured by culturing the cells at 37°C and in the presence ofsucrose (Fig. S6C).

To expand the utility of the two-plasmid system, we tested the editing efficiency ofthe system in two clinically isolated K. pneumoniae strains, KP_3744 and KP_5573. Theediting efficiencies of three different genes in the KP_3744 strain and four differentgenes in the KP_5573 strain were systematically investigated. The deletion efficienciesof all the genes tested in the KP_3744 strain (pyrF [Fig. 4A], fepB [Fig. 4B], and ramA [Fig.S7A]) and KP_5573 strain (fosA [Fig. 4C], pyrF [Fig. S7B], fepB [Fig. S7C], and ramA [Fig.4D]) were 100% (28–30). In addition to PCR screening and sequencing, we used thegrowth defect assay and the fosfomycin resistance assay to verify the deletions of pyrFand fosA, respectively. The cells lacking the pyrF gene (encoding orotidine 5-phosphatedecarboxylase) have a growth defect in uracil-free synthetic chemically defined me-

FIG 3 Scheme of the procedures for the iterative editing of the pCasKP-pSGKP system. For new rounds of genome editing, the spacer-introduced pSGKP-kmplasmid can be recycled by cultivation in the presence of sucrose. After all the desired editing, both plasmids can be cured by culturing the cells at 37°C andin the presence of sucrose. Apr, apramycin; Km, kanamycin.

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dium (CDM) (31). Disruption of the fosA gene (encoding dimeric Mn2�- and K�-dependent glutathione S-transferase) renders the cells more susceptive to fosfomycin.

Development of single-plasmid system pBECKP for base editing. The CRISPR-Cas9-mediated genome-editing method generates a DSB and requires a donor repairtemplate for editing. We sought to further simplify the editing process by developinga base editor in K. pneumoniae (Fig. 5A). The base editor directly mutates the target sitewithout generating a DSB or using a repair template. The cytidine base editor has thepotential to inactivate genes via converting four codons (CAA, CAG, CGA, and TGG) intopremature stop codons in a programmable manner. To harness the cytidine base editorfor base editing in K. pneumoniae, we constructed a single-plasmid editing system,pBECKP (Fig. 5B). The low-copy-number pBECKP plasmid expressed the sgRNA undercontrol of the J23119 promoter and the fusion protein of Cas9 nickase (nSpCas9, D10A)and rat APOBEC1 (rAPOBEC1) deaminase with a 16-residue XTEN linker under thecontrol of a weak promoter (32). Two BsaI sites and the sacB gene were introduced intothe plasmid for convenient spacer assembly and plasmid curing, respectively.

To assess the capacity of the pBECKP system for base editing in K. pneumoniae, wetransformed a fosA spacer-introduced pBECKP-km plasmid into the clinically isolatedKP_5573 strain. The fosA spacer contained a potentially editable TC7C8 motif. The C¡Tconversions of either or both the Cs at the positions of 7 and 8 could result in apremature stop codon in the fosA gene. As shown in Fig. 5C, both the Cs at positions

FIG 4 The two-plasmid pCasKP-pSGKP system allowed for highly efficient genome editing in the clinically isolated K. pneumoniae strains. (A) Deletion of thepyrF gene in the KP_3744 strain. The editing efficiency was 10/10. Lane CK, PCR band from the wild-type strain. The growth defect on the synthetic CDM platecontaining no uracil indicated disruption of the pyrF gene. (B) Deletion of the fepB gene in the KP_3744 strain. The editing efficiency was 10/10. (C) Deletionof the fosA gene in the KP_5573 strain. The editing efficiency was 10/10. The deletion of the fosA gene was confirmed by both the PCR and the tablet diffusionassay. (D) Deletion of the ramA gene in the KP_5573 strain. The editing efficiency was 10/10.

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7 and 8 were successfully mutated to Ts with 100% efficiencies in all the picked 8colonies. The high base-editing efficiency of the fosA gene was also observed in theindustrial KP_1.6366 strain (Fig. S8). In addition, we tested the capability of the pBECKPsystem for editing C-rich regions in the genome. Two C-rich spacers within the dhaKgene were assembled individually into the pBECKP-km plasmid. The plasmids weretransformed individually into the KP_1.6366 strain. As shown in Fig. 5D and S9, variousediting products with the conversions of Cs at different positions were obtained. Theseresults demonstrated that the pBECKP system could efficiently convert C to T in avariety of K. pneumoniae strains.

The human BE3 base editor has a strong cytidine deamination capacity within themutational spectra from positions 4 to 8 (termed the activity window) (16). Within theactivity window, the base-editing system has a high C-to-T conversion efficiency.Outside the activity window, the base editing could be detected occasionally, but theconversion efficiency was reduced drastically. Because the activity window of a cytidinebase editor may not be identical in different species, we systematically examined theactivity window and the sequence context preference of the pBECKP system in K.pneumoniae. Ten distinct spacers containing Cs at different positions were assembledinto the pBECKP plasmid. The plasmids were transformed into the KP_1.6366 strain. As

FIG 5 The pBECKP system enabled highly efficient base editing in K. pneumoniae. (A) Scheme of the procedures of pBECKP-mediated baseediting. The Cas9 nickase cleaves the nonedited strand and the APOBEC1 deaminase catalyzes the conversion of C to U. The resulting U:Gheteroduplex can be permanently converted to the T·A base pair by DNA repair or replication. (B) Map of the pBECKP-km plasmid. ThepBECKP-km plasmid contains the rAPOBEC1-XTEN-Cas9(D10A) fusion gene, the sgRNA expression cassette, the sacB gene, and thecopy-number-limiting gene rop. (C) W92 of the fosA gene in the KP_5573 strain was successfully mutated to a stop codon with anefficiency of 8/8 using the pBECKP system. A representative sequencing chromatogram for the fosA mutant is shown. The similarfosfomycin inhibition zone diameters between the deletion mutant strain and two point mutation strains indicated the successfuldisruption of the fosA gene. (D) Alignments of the editing products of a C-rich locus by the pBECKP system. The mutated Ts are coloredred. The Cs at different positions were mutated to Ts with different efficiencies by the pBECKP system.

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shown in Fig. 6, the editing efficiency of TC was higher than that of CC and AC. GC hadthe lowest editing efficiency, consistent with the sequence context preference of themammalian base editor (16). The Cs from the TC motif at positions 3 to 8 wereconverted to Ts with efficiencies of almost 100%, whereas the editing efficiencies of theCs at other positions were much lower, indicating that the activity window of thepBECKP system was from positions 3 to 8. Intriguingly, a few Cs at position 9 of spacer8 and position 7 of spacer 10 were mutated to As but not Ts (Fig. 6). The editingby-product was also observed in the editing process of eukaryotic base editors (33–35).

Dissection of drug resistance mechanisms using the two editing systems. Thequick dissemination of carbapenem-resistant K. pneumoniae has posed a severe threatto public health worldwide. The mobile genetic elements encoding carbapenemasesdramatically accelerate the global expansion of carbapenem resistance. The acquirablecarbapenemases are largely divided into the KPC, NDM, OXA-48, VIM, and IMP types(36). These carbapenemases are often coproduced with extended-spectrum beta-lactamases (ESBLs) in clinically isolated carbapenem-resistant K. pneumoniae. We usedboth the pCasKP-pSGKP and pBECKP systems to verify the contribution of carbapen-emases in carbapenem resistance.

First, we sought to delete the genes encoding carbapenemases and ESBLs individ-ually in a hypermucoviscous carbapenem-resistant K. pneumoniae strain, KP_CRE23,using the pCasKP-pSGKP system. The KP_CRE23 strain harbored one carbapenemasegene, blaKPC-2, and two ESBL genes, blaSHV and blaCTX-M-65 (37). Because the KP_CRE23strain is resistant to kanamycin, the kanamycin marker in both the pSGKP-km and thepBECKP-km plasmids was replaced with the spectinomycin marker, resulting in thepSGKP-spe and the pBECKP-spe plasmids. As shown in Fig. 7A, we obtained the desiredchromosomal blaSHV deletion mutant with an efficiency of 4/12. However, in the case

FIG 6 Determination of the activity window and sequence context preference of the pBECKP system in K. pneumoniae. TheCs with high editing efficiencies were marked with red squares. A few Cs at position 9 of spacer8 and position 7 of spacer10were mutated to As but not Ts. These sites were colored green.

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of the deletions of the plasmid-borne blaKPC-2 and blaCTX-M-65 genes using the samemethod, neither the desired gene deletion bands nor the wild-type bands wereamplified by PCR in all the tested colonies (Fig. S10). A possible reason is that in theabsence of selection pressure, the DSB of the blaKPC-2 gene- and the blaCTX-M-65

gene-carrying plasmids led to the plasmid removal without repair. It has been reportedthat Cas9 nuclease-mediated DSB on a plasmid can be used for plasmid removal inGram-negative bacteria (38).

Next, we attempted to use the base editor pBECKP-spe to inactivate the blaKPC-2 andblaCTX-M-65 genes, because the pBECKP-spe system executed editing by the introduc-tion of a single-stranded DNA break instead of a DSB. As shown in Fig. 7B and C, theblaKPC-2 and blaCTX-M-65 genes were successfully mutated, resulting in the introductionof premature stop codons. The editing efficiencies were 8/8 and 2/8, respectively. Wethen examined the imipenem (a major carbapenem class drug) susceptibilities of thewild-type KP_CRE23 strain and three mutant strains using the inhibition zone and MICassays. As shown in Fig. 7D, inactivation of the blaKPC-2 gene drastically increased thebacterial susceptibility to imipenem, whereas no significant drug susceptibility differ-ence was observed when the blaSHV gene was deleted or the blaCTX-M-65 gene wasinactivated. These results verified that the blaKPC-2 gene was the key factor thatcontributed to carbapenem resistance in the hypermucoviscous K. pneumoniae strainKP_CRE23. These approaches can be applied to a more complex system to dissect thedrug resistance mechanisms of K. pneumoniae.

DISCUSSION

Klebsiella pneumoniae is an important industrial microorganism and human patho-gen, but traditional genetic manipulation in K. pneumoniae is often time-consumingand laborious. Therefore, more efficient and simple genetic tools are highly desirable.In this study, by harnessing the powerful DNA-cleaving ability of the engineeredCRISPR-Cas9 system and the strong recombination capacity of the lambda Red system,we have developed a convenient and efficient two-plasmid system, pCasKP-pSGKP, foriterative and scarless chromosomal gene deletion and insertion in K. pneumoniae. Wefirst constructed a single-plasmid CRISPR-Cas9 system which could efficiently cleave thegenomic DNA of K. pneumoniae. Due to the lack of the nonhomologous end-joiningpathway, the DSB created by Cas9 nuclease on the chromosome was lethal to K.pneumoniae. Although the repair templates had been supplied by cloning them intothe single CRISPR-Cas9 plasmid, no desirable deletion mutants were obtained, indicat-

FIG 7 blaKPC-2 is the key factor for carbapenem resistance in multidrug-resistant hypermucoviscous K. pneumoniae strain KP_CRE23. (A)pCasKP-pSGKP-mediated deletion of the chromosomal blaSHV gene. The deletion efficiency was 4/12. (B and C) W164 of the plasmid-borneblaKPC-2 gene (B) and Q136 of the plasmid-borne blaCTX-M-65 gene (C) were successfully mutated to stop codons with efficiencies of 8/8and 2/8, respectively, by the pBECKP system. (D) blaKPC-2 was the key gene for carbapenem resistance in K. pneumoniae strain KP_CRE23.

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ing the weak capacity of the native homology-directed repair system of K. pneumoniae.To repair the DSB on the chromosome created by the Cas9 nuclease, we introduced thelambda Red recombination system, which efficiently repaired the cleaved genomicDNA with the utilization of any type of repair template, including ssDNA.

To further simplify the editing process, we have developed a highly efficient cytidinebase-editing system, pBECKP, by fusing a cytidine deaminase to the Cas9 nickase,enabling precise C¡T conversions in both the chromosome and the plasmids. Theactivity window and the preference of the adjacent base of the editable sites weresystematically investigated in K. pneumoniae. The pBECKP system could irreversiblyinactivate genes by converting four codons (CAA, CAG, CGA, and TGG) into prematurestop codons in a programmable manner. One major limitation of inactivating genes viathe pBECKP system arises from the limited PAM sites that are adjacent to the afore-mentioned four codons. The pBECKP requires the presence of a nearby NGG sitelocated 13 to 18 bp away from an editable CAA, CAG, CGA, or TGG codon. The recentlyevolved xCas9 protein that recognizes a broad range of PAM sequences, including NG,GAA, and GAT (39), may expand the base-editing scope of the pBECKP system.

The pCasKP-pSGKP system and the pBECKP system have editing efficiencies in avariety of clinically isolated K. pneumoniae strains. By using both editing systems, weverified the carbapenem resistance mechanism of the multidrug-resistant hypermuco-viscous K. pneumoniae strain KP_CRE23. Because the KP_CRE23 strain was resistant tokanamycin, the kanamycin marker in both the pSGKP-km and pBECKP-km plasmids wasreplaced with the spectinomycin marker. Given that some K. pneumoniae isolates wereinsensitive to apramycin and/or kanamycin, the antibiotic resistances may limit theapplications of the pCasKP-pSGKP system and the pBECKP system for genetic manip-ulations in multidrug-resistant K. pneumoniae strains. By testing the drug sensitivity ofseveral clinically isolated K. pneumoniae strains, we detected that the majority of themwere sensitive to hygromycin B. Thereby, we constructed a new plasmid, pCasKP-hph,which could serve as an alternative option for genetic manipulation in thoseapramycin-resistant K. pneumoniae strains. The antibiotic marker could also be replacedwith any other suitable selection marker for editing in different K. pneumoniae strains.

The off-target effect was rarely noticed for DSB-based genome editing in NHEJ-deficient bacteria, because the cells with off-target events cannot survive. However,because the base editor directly mutated the target site without generating a DSB,potential off-target effects were not lethal to the cells edited by pBECKP system. Toobtain a high editing efficiency and reduce potential off-target effects, the spacers usedin this study were designed using the sgRNAcas9 software (40). The sgRNAcas9software could screen all the suitable spacer sequences in the target genes andevaluate their potential off-target sites throughout the K. pneumoniae genome.

Overall, we have engineered the two-plasmid system pCasKP-pSGKP for genomeediting and the single-plasmid system pBECKP for base editing in a variety of K.pneumoniae strains. Given the simple construction procedures and high efficiency,future applications of the two editing systems should dramatically facilitate a widevariety of investigations, such as gene characterization, drug discovery, and metabolicengineering, in K. pneumoniae and relevant Enterobacteriaceae species.

MATERIALS AND METHODSPlasmids, bacterial strains, primers, and growth conditions. All of the plasmids used in this study

are listed in Table 1, and all of the bacterial strains used in this study are listed in Table 2. The primersused in this study were purchased from Genewiz (Suzhou, China) and are listed in Table S1. Escherichiacoli DH5� and K. pneumoniae strains were grown in lysogeny broth (LB) medium (per liter, 5 g of yeastextract, 10 g of tryptone, 10 g of NaCl [pH 7.2 to �7.4]). Antibiotics were added at the followingconcentrations: 30 to 50 �g/ml apramycin, 100 �g/ml hygromycin B, 50 �g/ml kanamycin, and 50 to 100�g/ml spectinomycin for both the E. coli and K. pneumoniae strains.

Plasmid construction. The temperature-sensitive pCasKP-apr plasmid was constructed using thefollowing procedures. The rpsL promoter was PCR amplified from the genomic DNA of the K. pneumoniaestrain KP_1.6366. The gene encoding the Cas9 nuclease was amplified from the pCasSA plasmid (41). Theaforementioned two fragments along with the NdeI_linearized pKOBEG-apr plasmid (23) were assembledtogether using In-Fusion cloning, resulting in the final plasmid pCasKP-apr. The pCasKP-hph plasmid was

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constructed by replacing the apramycin resistance gene of the pCasKP-apr plasmid with the hygromycinB resistance gene.

The sgRNA expression cassette was synthesized commercially by Genewiz (Suzhou, China). Thecassette contained the following three elements: the constitutive J23119 promoter, two BsaI restrictionsites for the insertion of a 20-bp spacer, and the sgRNA scaffold. The sgRNA expression cassette wascloned into the EcoRV-digested pUC57 vector, yielding the pUC57-sgRNA plasmid. Then, the sacB geneamplified from the pCasPA plasmid (42) was inserted into the HindIII-digested pUC57-sgRNA plasmid viaIn-Fusion cloning, resulting in the final PSGKP-km plasmid. The pSGKP-spe plasmid was constructed byreplacing the kanamycin resistance gene of the pSGKP-km plasmid with the spectinomycin resistancegene.

The pBECKP-km plasmid was constructed with the following procedure. The low-copy-numberplasmid backbone containing pBR322_origin, the rop gene, and the kanamycin resistance marker wereamplified from the pET28a plasmid. The sgRNA expression cassette and the sacB gene were amplifiedfrom the pSGKP-km plasmid. The two fragments were assembled into a plasmid by In-Fusion cloning.Finally, the rAPOBEC1-XTEN-Cas9(D10A) cassette amplified from the pnCasSA-BEC plasmid (32) wasinserted into the HindIII site of the aforementioned plasmid to form the final all-in-one pBECKP-kmplasmid. The pBECKP-spe plasmid was constructed by replacing the kanamycin resistance gene of thepBECKP-km plasmid with the spectinomycin resistance gene.

Preparation of competent cells and electroporation. For the K. pneumoniae wild-type strain, 1 mlovernight culture from a fresh single colony was diluted into 100 ml of LB broth and incubated at 37°C.

TABLE 1 Plasmids used in this study

Plasmid Descriptiona Reference or source

pCasSA Plasmid carrying bacterial Cas9 nuclease gene (Kmr Cmr) 41pKOBEG-apr Thermosensitive plasmid, carries lambda Red genes (Aprr) 23pOSCAR Cloning plasmid (Sper) 43pCasPA Plasmid carrying sacB gene (Tetr) 42pnCasSA-BEC Plasmid carrying rAPOBEC1-XTEN-Cas9(D10A) gene (Kmr Cmr) 32pET28a Low-copy-no. plasmid (Kmr) Lab stockpMD19-mcherry Plasmid carrying mcherry gene (Ampr) Lab stockpMD19-hyg Plasmid carrying hph gene (Ampr Hygr) Lab stockpCas-sgRNAKP K. pneumoniae single-plasmid CRISPR-Cas9 editing vector (Aprr) This studypCas-sgRNAKP_dhaF pCas-sgRNAKP derivative with dhaF spacer This studypCas-sgRNAKP_dhaF_HR pCas-sgRNAKP derivative with dhaF spacer and �1 kb each repair arm This studypCasKP-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 inK. pneumoniae (Hygr)

This study

pSGKP-km Plasmid expressing sgRNA in K. pneumoniae (Kmr) This studypSGKP-spe Plasmid expressing sgRNA in K. pneumoniae (Sper) This studypSGKP_dhaF pSGKP-km derivative with dhaF spacer This studypSGKP_dhaF_HR pSGKP-km derivative with dhaF spacer and �1 kb each repair arm This studypSGKP_dhaK pSGKP-km derivative with dhaK spacer This studypSGKP_dhaK_HR pSGKP-km derivative with dhaF spacer and �0.5 kb each repair arm This studypSGKP_fosA pSGKP-km derivative with fosA spacer This studypSGKP_pyrF pSGKP-km derivative with pyrF spacer This studypSGKP_fepB pSGKP-km derivative with fepB spacer This studypSGKP_ramA pSGKP-km derivative with ramA spacer This studypSGKP-spe_blaKPC pSGKP-spe derivative with blaKPC spacer This studypSGKP-spe_blaSHV pSGKP-spe derivative with blaSHV spacer This studypSGKP-spe_blaCTX pSGKP-spe derivative with blaCTX spacer This studypBECKP-km K. pneumoniae base editing vector (Kmr) This studypBECKP-spe K. pneumoniae base editing vector (Sper) This studypBECKP_fosA_1 pBECKP-km derivative with fosA spacer 1 This studypBECKP_fosA_2 pBECKP-km derivative with fosA spacer 2 This studypBECKP_fosA_3 pBECKP-km derivative with fosA spacer 3 This studypBECKP_dhaK_1 pBECKP-km derivative with dhaK spacer 1 This studypBECKP_dhaK_2 pBECKP-km derivative with dhaK spacer 2 This studypBECKP_dhaK_3 pBECKP-km derivative with dhaK spacer 3 This studypBECKP_dhaK_4 pBECKP-km derivative with dhaK spacer 4 This studypBECKP_dhaF_1 pBECKP-km derivative with dhaF spacer 1 This studypBECKP_dhaF_2 pBECKP-km derivative with dhaF spacer 2 This studypBECKP_dhaF_3 pBECKP-km derivative with dhaF spacer 3 This studypBECKP_dhaF_4 pBECKP-km derivative with dhaF spacer 4 This studypBECKP_dhaF_5 pBECKP-km derivative with dhaF spacer 5 This studypBECKP-spe_blaSHV pBECKP-spe derivative with blaSHV spacer This studypBECKP-spe_blaCTX pBECKP-spe derivative with blaCTX spacer This studyaKmr, kanamycin resistance; Cmr, chloramphenicol resistance; Aprr, apramycin resistance; Sper, spectinomycin resistance; Tetr, tetracycline resistance; Ampr, ampicillinresistance; Hygr, hygromycin B resistance.

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When the optical density at 600 nm (OD600) of the cell culture reached 0.5 to 0.7, the culture wasimmediately chilled on ice for 20 min and then harvested by centrifugation at 7,200 � g for 5 min. Thesupernatant was discarded, and the cells were resuspended by pipetting gently with 15 ml of sterileice-cold 10% glycerol. The centrifugation and resuspension steps were repeated twice. Finally, the cellswere resuspended with 1 ml of ice-cold 10% glycerol. Fifty-microliter aliquots were frozen in liquidnitrogen and stored at �80°C.

For the pCasKP-harboring K. pneumoniae strain, 1 ml of overnight culture from a fresh single colonywas diluted into 100 ml of LB broth containing 30 �g/ml apramycin and incubated at 30°C. When thecell density reached an OD600 of approximately 0.2, 1 ml of 20% L-arabinose was added for induction ofthe lambda Red recombineering operon of pCasKP. After induction at 30°C for 2 h, the culture wasprepared as electrocompetent cells in a way similar to that of the wild-type K. pneumoniae.

For electroporation, 50 �l of electrocompetent cells was thawed on ice for several minutes. Then, thecells were mixed with no more than 5 �l plasmid or donor template. The mixture was transferred intoa 2-mm electroporation cuvette (Bio-Rad) and electroporated at 2.5 kV, 200 �, and 25 �F. After beingpulsed, the cells were recovered in 1 ml antibiotic-free LB broth and incubated at 30°C for 1.5 h beforebeing plated onto LB agar plates supplemented with the required antibiotics. The plates were incubatedat 30°C overnight.

Genome editing and base editing. The detailed protocols for spacer cloning, genome editing, baseediting, and plasmid curing in K. pneumoniae are provided in the supplemental material.

Antimicrobial susceptibility assay. For the inhibition testing, a fresh K. pneumoniae suspension wasadjusted to a 0.5 McFarland turbidity standard and then diluted 10 times with saline. The dilutedbacterial suspension was evenly coated onto a Mueller-Hinton (MH) agar plate. The plate was dried for5 min. A fosfomycin (50 �g/tablet; Oxoid) or imipenem (10 �g/tablet; Oxoid) tablet was placed in thecenter of the aforementioned MH plate. The plate was incubated at 35°C for 20 h to produce theinhibition zones.

For the MIC assay, the MICs of imipenem for the carbapenem-resistant KP_CRE23 strain and threemutant strains were determined using the 96-well broth microdilution method recommended by theClinical and Laboratory Standards Institute (45). In brief, a fresh K. pneumoniae suspension was adjustedto a 0.5 McFarland turbidity standard and then diluted 10 times with saline. The 2-�l diluted solutionswith 5.0 � 106 CFU bacterial cells were inoculated into 200 �l MH liquid medium containing serial 2-folddilution concentrations of imipenem (0.5 to �64 �g/ml). The imipenem-free MH liquid medium was usedas the control. After incubation at 35°C for 20 h, the MICs of complete growth inhibition were determinedby visual inspection.

Data availability. All the plasmids constructed in this study were validated by PCR, enzymedigestion, and DNA sequencing. Their sequences were submitted to the GenBank database under theaccession numbers MH587683 (pSGKP-km), MH587684 (pSGKP-spe), MH587685 (pBECKP-km), MH587686(pBECKP-spe), MH587687 (pCasKP-apr), and MH587688 (pCasKP-hph). All the plasmids constructed in this

TABLE 2 Bacterial strains used in this study

Strain Description or genotype Reference

E. coli DH5� F� �80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK�, mK

�)phoA supE44 �� thi-1 gyrA96 relA1

Lab stock

K. pneumoniaeKP_1.6366 Wild-type industrial K. pneumoniae strain 44KP_1.6366 dhaF KP_1.6366 ΔdhaF This studyKP_1.6366 dhaK KP_1.6366 ΔdhaK This studyKP_1.6366 fosA KP_1.6366 ΔfosA This studyKP_1.6366 fosA::mcherry KP_1.6366 ΔfosA::mcherry This studyKP_1.6366 dhaF dhaK KP_1.6366 ΔdhaF ΔdhaK This studyKP_1.6366 fosA W92 to stop KP_1.6366 fosA W92 mutation to stop codon This studyKP_3744 Wild-type clinically isolated K. pneumoniae strain Lab stockKP_3744 pyrF KP_3744 ΔpyrF This studyKP_3744 fepB KP_3744 ΔfepB This studyKP_3744 ramA KP_3744 ΔramA This studyKP_5573 Wild-type clinically isolated K. pneumoniae strain Lab stockKP_5573 fosA KP_5573 ΔfosA This studyKP_5573 pyrF KP_5573 ΔpyrF This studyKP_5573 fepB KP_5573 ΔfepB This studyKP_5573 ramA KP_5573 ΔramA This studyKP_5573 fosA W92 to stop KP_5573 fosA W92 mutation to stop codon This studyKP_CRE23 Wild-type clinically isolated K. pneumoniae strain with multidrug

resistance and hypermucoviscosity37

KP_CRE23 blaSHV KP_CRE23 ΔblaSHV This studyKP_CRE23 blaKPC-2 W164 to stop KP_CRE23 blaKPC-2 W164 mutation to stop codon This studyKP_CRE23 blaCTX-M-65 Q136 tostop

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

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study will be available in Addgene with the numbers 117231 (pCasKP-apr), 117232 (pCasKP-hph), 117233(pSGKP-km), 117234 (pSGKP-spe), 117235 (pBECKP-km), and 117236 (pBECKP-spe).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01834-18.

SUPPLEMENTAL FILE 1, PDF file, 1.8 MB.

ACKNOWLEDGMENTSWe thank Jian Hao from the Shanghai Advanced Research Institute, Chinese Acad-

emy of Sciences, for generously providing the K. pneumoniae KP_1.6366 strain.This work was financially supported by the National Key R&D Program of China

(grant 2017YFA0506800), the National Natural Science Foundation of China (grants91753127 and 31700123), the Shanghai Committee of Science and Technology, China(grant 17ZR1449200), the ShanghaiTech Startup Funding, and the “Young 1000 Talents”Program to Q.J.; the China Postdoctoral Science Foundation (grant 2018M632190) toY.W.; and the Shanghai Sailing Program (grant 18YF1416500) to W.C.

Two patent applications have been submitted for the two-plasmid genome-editingsystem pCasKP-pSGKP and the single-plasmid base-editing system pBECKP.

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