Genetics is a key means to study bacterial physiology. However, the highly desirable scarless genetic manipulation is often time-consuming and laborious for the major human pathogen K. pneumoniae. We developed a CRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system, enabling rapid, highly efficient, and iterative genome editing in both industrial and clinically isolated K. pneumoniae strains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistant K. pneumoniae strain, elucidating that the blaKPC-2 gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant K. pneumoniae strain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverse K. pneumoniae strains and relevant Enterobacteriaceae species, such as gene characterization, drug discovery, and metabolic engineering.
KEYWORDS: CRISPR, Cas9, Klebsiella pneumoniae, genetic engineering, genome editing, base editing
ABSTRACT
Klebsiella pneumoniae is a promising industrial microorganism as well as a major human pathogen. The recent emergence of carbapenem-resistant K. pneumoniae has posed a serious threat to public health worldwide, emphasizing a 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 desirable scarless genetic manipulation in K. pneumoniae, is often time-consuming and laborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for precise and iterative genome editing in K. pneumoniae. By harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome cleavage system and the lambda Red recombination system, pCasKP-pSGKP enabled highly efficient genome editing in K. pneumoniae using a short repair template. Moreover, we developed a cytidine base-editing system, pBECKP, for precise C→T conversion in both the chromosomal and plasmid-borne genes by engineering the fusion of the cytidine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP-pSGKP and the pBECKP tools, the blaKPC-2 gene was confirmed to be the major factor that contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistant K. pneumoniae strain. The development of the two editing tools 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-consuming and laborious for the major human pathogen K. pneumoniae. We developed a CRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system, enabling rapid, highly efficient, and iterative genome editing in both industrial and clinically isolated K. pneumoniae strains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistant K. pneumoniae strain, elucidating that the blaKPC-2 gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant K. pneumoniae strain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverse K. pneumoniae strains and relevant Enterobacteriaceae species, such as gene characterization, drug discovery, and metabolic engineering.
INTRODUCTION
Klebsiella pneumoniae is a high-GC-content Gram-negative bacillus of the Enterobacteriaceae family and is widely distributed in the natural environment and on the mucosal surfaces of mammals. It is considered a promising industrial microorganism because 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 of hospital- and community-acquired infections, such as pneumonia, bacteremia, and urinary tract infections (2). In recent years, the emergence of hypermucoviscous and multidrug-resistant K. pneumoniae strains, in particular, carbapenem-resistant hypermucoviscous K. pneumoniae strains, has posed a severe public health crisis worldwide (3–5). Thereby, novel therapeutic means against multidrug-resistant K. pneumoniae infections are urgently needed.
The development of novel therapeutic means against drug-resistant K. pneumoniae infections would benefit greatly from efficient and convenient genome editing and screening tools, which allow effective identification of the key genes and pathways responsible for bacterial virulence and drug resistance. Although the lambda Red recombination system has been developed and widely utilized for genetic manipulation and the clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) system has been developed recently for transcriptional inhibition in K. pneumoniae, the highly desirable scarless and precise genome editing in K. pneumoniae is still time-consuming and laborious (6, 7). For instance, to construct a markerless deletion mutant in K. pneumoniae, a target gene is first replaced by an antibiotic marker via a double-crossover homologous recombination process mediated by the lambda Red recombination proteins (Gam, Bet, and Exo). Second, the antibiotic marker is eliminated by the utilization of a helper plasmid expressing the FLP recombinase (FRT). The FLP recombinase directly binds to the repeated FLP recognition sites flanking the antibiotic gene and catalyzes the elimination reaction, leaving an FRT scar in the place of the target gene. When multiple rounds of genetic modification are performed using the aforementioned method, the introduction of multiple FRT scars in the genome may lead to genome instability by causing genome rearrangement (8).
The recently discovered CRISPR-Cas9 system allows for the efficient generation of a double-strand break (DSB) at a desired site of the target genome (9, 10), thereby raising the possibility of one-step scarless genome editing in K. pneumoniae. The CRISPR system is an adaptive immune system and is utilized by bacteria and archaea to fight against invading phages and foreign plasmids (11, 12). The wildly utilized CRISPR-Cas9 system is composed of two components, the Cas9 nuclease from Streptococcus pyogenes and a single artificial chimeric guide RNA (sgRNA) (13). The sgRNA directs the Cas9 protein to a target genomic locus through complementary base pairing to a target sequence 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 most bacteria, including Klebsiella pneumoniae, chromosomal cleavage is lethal to bacterial cells unless it is repaired by the homologous recombination (HR) pathway with the utilization of exogenously supplied donor DNA repair templates (15). Thereby, precise genetic manipulation, including gene deletions, point mutations, and gene insertions, can be achieved by simply customizing an approximately 20-nucleotide (nt) spacer sequence and a designed donor repair template.
Furthermore, the recently developed CRISPR RNA-guided deaminase systems enable precise 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 the adenosine editor ABE (18). Each base editor is composed of a Cas9 nickase (D10A in the case of S. pyogenes Cas9 [SpCas9]) or a dead Cas9 protein (D10A and H840A in the case of SpCas9) and a deaminase fused to the Cas9 protein. Relying on the base pairing between a target sequence and the 20-nt guide RNA sequence, the tethered deaminase can be directed to any target locus to perform nucleoside deamination through a deamination reaction (C→U for the BEC editor and A→I for the ABE editor). In living cells, the DNA repair or replication mechanism would efficiently convert the U:G or I:T 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 nucleosides without the formation of DSB or the utilization of a donor template.
In this study, we developed a CRISPR-Cas9-mediated genome-editing method and a base-editing system enabling rapid, highly efficient, and iterative genome editing in both 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 contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistant K. pneumoniae strain. The development of these genome-editing tools will dramatically accelerate a wide variety of investigations in K. pneumoniae.
RESULTS
Establishment of a single-plasmid CRISPR-Cas9 system in K. pneumoniae.
To develop a convenient and scarless genetic manipulation method in K. pneumoniae, we first sought to harness the CRISPR-Cas9 system for genome editing. To access the functionality of the CRISPR-Cas9 system in K. pneumoniae, we constructed a single-plasmid system, pCas9-sgRNAKP, that expressed both the well-studied Streptococcus pyogenes Cas9 protein and the sgRNA in the same plasmid (19). The transformation of the 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 effective cleavage of bacterial genome by the CRISPR-Cas9 system.
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). Genome editing 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 plasmid was transformed into the KP_1.6366 strain as a control (left).
Next, we assembled the repair templates (∼1 kb each) of the dhaF gene into the dhaF spacer-introduced pCas9-sgRNAKP plasmid to test the functionality of the system for gene deletion in K. pneumoniae. The transformation of the assembled plasmid into K. pneumoniae yielded only fewer than 5 colonies (Fig. 1). Further PCR screening analysis revealed that none of them were the desired deletion mutants, indicating that the intrinsic homologous recombination capacity of K. pneumoniae was not great enough for the direct repair of the lethiferous double-stranded DNA break of the genome.
To alleviate the toxicity of chromosomal cleavage by the Cas9 nuclease, two versions of Cas9 nickase expression plasmids, pnCas9D10A_sgRNAKP and pnCas9H840A_sgRNAKP, were constructed by mutating the active sites of Cas9 protein Aps10 or His840 to Ala, respectively. The transformations of the Cas9 nickase plasmids containing both the dhaF spacer and the corresponding repair template (∼1 kb each) indeed yielded plenty of colonies. However, PCR screening and further sequencing revealed that no desired homologous recombination-repair events were observed (see Fig. S1 in the supplemental material). It is likely that K. pneumoniae preferred to accurately repair the DNA nick using the complementary strand, rather than 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 stronger recombination capacity than that of normal bacterial cells (20, 21). We sought to increase the homologous recombination capacity of K. pneumoniae by introducing the phage lambda Red recombination system into the bacteria. To achieve this, we designed and constructed a two-plasmid system, pCasKP-pSGKP (Fig. 2A and B). The pCasKP plasmid expressed the Cas9 protein under the control of the constitutive K. pneumoniae rpsL promoter and the lambda Red recombination proteins (Gam, Bet, and Exo) under the control of an l-arabinose-inducible ParaB promoter. The pSGKP plasmid expressed the sgRNA under the control of the synthetic constitutive J23119 promoter (22). Two reversed BsaI sites were inserted between the J23119 promoter and the sgRNA scaffold for the seamless and one-step assembly of spacers (Fig. S2). In addition, the temperature-sensitive replicon repA101(Ts) (23) and the sucrose-sensitive gene sacB (24) were introduced into the pCasKP and pSGKP plasmids, respectively, for easy plasmid curing after editing.
FIG 2.
Genome editing in K. pneumoniae using a two-plasmid pCasKP-pSGKP system. (A) Scheme for the CRISPR-Cas9 and lambda Red recombination-mediated genome-editing method. The sgRNA-Cas9 complex cleaves the double-strand DNA proximal to a PAM site, generating a double-stranded DNA break. The double-stranded DNA break is repaired via lambda Red-mediated homologous recombination using a donor template. (B) Maps of the pCasKP-apr and pSGKP-km plasmids. pCasKP-apr contains the Cas9 gene with a constitutive rpsL promoter, the lambda Red recombination genes (gam, bet, and exo) with an l-arabinose-inducible promoter ParaB, and the temperature-sensitive replicon repA101(Ts) (repA101ts). pSGKP-km contains the sgRNA with the synthetic J23119 promoter 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 templates in the KP_1.6366 strain. Two hundred nanograms of dhaK spacer-introduced pSGKP_dhaK plasmid, 300 ng pSGKP_dhaK_HR plasmid containing the repair template (∼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 μM ssDNA (90 nt) were used for the transformations shown from left to right, respectively. Error bars represent standard deviation from the results from three independent experiments.
To assess the genome-editing ability of the constructed two-plasmid system, we sought to delete the dhaF gene in the industrial K. pneumoniae strain KP_1.6366. To do this, 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 the induction of l-arabinose, the cells containing the pCasKP-apr plasmid were collected and prepared as the competent cells. Then, the dhaF deletion plasmid pSGKP-dhaF-HR was transformed into the aforementioned pCasKP-apr-harboring competent cells by electroporation. The pSGKP-dhaF-HR plasmid was constructed by assembling both the dhaF spacer and the repair arms of the dhaF gene (∼1 kb each) into the pSGKP-km plasmid. The subsequent transformation yielded >1,000 colonies. Twenty colonies were randomly picked to test the editing efficiency. As shown in Fig. 2C, successful deletion of the dhaF gene was confirmed in all the picked colonies by both PCR and sequencing.
To simplify the plasmid construction procedures and accelerate the genome-editing process, we attempted to utilize the linear homologous DNA fragment as the repair template. The lambda Red recombination system is capable of using the plasmid-borne donor DNA, linear double-stranded DNA (dsDNA), or single-stranded DNA (ssDNA) as the repair template (25). To evaluate the editing efficiency of linear repair templates, a linear dsDNA (∼500 bp each) and an ssDNA (45 nt each) were cotransformed individually with the dhaK spacer-introduced pSGKP-km plasmid (pSGKP_dhaK) into the l-arabinose-induced pCasKP-apr-harboring cells to delete the dhaK gene (26). The transformations of the pSGKP_dhaK plasmid (only dhaK spacer) and the pSGKP_dhaK_HR plasmid (dhaK spacer and assembled with ∼500 bp each repair template) were used as the negative and positive controls, respectively. As shown in Fig. 2D and S3A, >1,000 colonies were observed for all the transformations containing any type of the donor repair templates, whereas fewer than 10 colonies were obtained for the transformation without a repair template. Further PCR screening and sequencing showed that the deletion efficiencies were 100% for all the transformations containing the repair templates (Fig. S3B to D). Moreover, we assessed the capacity of the two-plasmid system pCasKP-pSGKP to delete the fosA gene with the utilization of ssDNA as the repair 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 for gene insertion in K. pneumoniae. We attempted to replace the fosA gene with the mcherry 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 colonies were recovered, and the insertion efficiency was 9/10 (Fig. S5B). Together, these experiments demonstrated that the two-plasmid pCasKP-pSGKP system possessed a great capacity for genetic manipulation in K. pneumoniae with the utilization of a short repair template.
Complicated physiology study and metabolic engineering of K. pneumoniae require the genetic manipulation of multiple genes, thereby requiring multiple rounds of genome editing. For the second-round editing, the spacer-incorporated pSGKP-km plasmid needs to be recycled for different target loci, while the pCasKP-apr plasmid can be maintained to express the Cas9 protein and the lambda Red recombination system (Fig. 3). We inoculated one colony containing the desired dhaK deletion into lysogeny broth (LB) medium with the supplementation of apramycin. The cells were cultured at 30°C overnight. Next, a fraction of the cells was streaked onto an LB agar plate containing apramycin and sucrose and incubated at 30°C until colonies were visible. As shown in Fig. S6A, all the four randomly picked colonies could only grow normally on the plate containing apramycin, whereas none of them could grow on the plate containing both apramycin and kanamycin, confirming the successful removal of the pSGKP-km plasmid with the maintenance of the pCasKP-apr plasmid. Next, the dhaF 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-km plasmids could be easily cured by culturing the cells at 37°C and in the presence of sucrose (Fig. S6C).
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-km plasmid 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 and in the presence of sucrose. Apr, apramycin; Km, kanamycin.
To expand the utility of the two-plasmid system, we tested the editing efficiency of the system in two clinically isolated K. pneumoniae strains, KP_3744 and KP_5573. The editing efficiencies of three different genes in the KP_3744 strain and four different genes in the KP_5573 strain were systematically investigated. The deletion efficiencies of 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 the growth defect assay and the fosfomycin resistance assay to verify the deletions of pyrF and fosA, respectively. The cells lacking the pyrF gene (encoding orotidine 5-phosphate decarboxylase) have a growth defect in uracil-free synthetic chemically defined medium (CDM) (31). Disruption of the fosA gene (encoding dimeric Mn2+- and K+-dependent glutathione S-transferase) renders the cells more susceptive to fosfomycin.
FIG 4.
The two-plasmid pCasKP-pSGKP system allowed for highly efficient genome editing in the clinically isolated K. pneumoniae strains. (A) Deletion of the pyrF 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 plate containing 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) Deletion of 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 diffusion assay. (D) Deletion of the ramA gene in the KP_5573 strain. The editing efficiency was 10/10.
Development of single-plasmid system pBECKP for base editing.
The CRISPR-Cas9-mediated genome-editing method generates a DSB and requires a donor repair template for editing. We sought to further simplify the editing process by developing a base editor in K. pneumoniae (Fig. 5A). The base editor directly mutates the target site without generating a DSB or using a repair template. The cytidine base editor has the potential to inactivate genes via converting four codons (CAA, CAG, CGA, and TGG) into premature stop codons in a programmable manner. To harness the cytidine base editor for base editing in K. pneumoniae, we constructed a single-plasmid editing system, pBECKP (Fig. 5B). The low-copy-number pBECKP plasmid expressed the sgRNA under control 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 the control of a weak promoter (32). Two BsaI sites and the sacB gene were introduced into the plasmid for convenient spacer assembly and plasmid curing, respectively.
FIG 5.
The pBECKP system enabled highly efficient base editing in K. pneumoniae. (A) Scheme of the procedures of pBECKP-mediated base editing. The Cas9 nickase cleaves the nonedited strand and the APOBEC1 deaminase catalyzes the conversion of C to U. The resulting U:G heteroduplex can be permanently converted to the T·A base pair by DNA repair or replication. (B) Map of the pBECKP-km plasmid. The pBECKP-km plasmid contains the rAPOBEC1-XTEN-Cas9(D10A) fusion gene, the sgRNA expression cassette, the sacB gene, and the copy-number-limiting gene rop. (C) W92 of the fosA gene in the KP_5573 strain was successfully mutated to a stop codon with an efficiency of 8/8 using the pBECKP system. A representative sequencing chromatogram for the fosA mutant is shown. The similar fosfomycin inhibition zone diameters between the deletion mutant strain and two point mutation strains indicated the successful disruption of the fosA gene. (D) Alignments of the editing products of a C-rich locus by the pBECKP system. The mutated Ts are colored red. The Cs at different positions were mutated to Ts with different efficiencies by the pBECKP system.
To assess the capacity of the pBECKP system for base editing in K. pneumoniae, we transformed a fosA spacer-introduced pBECKP-km plasmid into the clinically isolated KP_5573 strain. The fosA spacer contained a potentially editable TC7C8 motif. The C→T conversions of either or both the Cs at the positions of 7 and 8 could result in a premature stop codon in the fosA gene. As shown in Fig. 5C, both the Cs at positions 7 and 8 were successfully mutated to Ts with 100% efficiencies in all the picked 8 colonies. The high base-editing efficiency of the fosA gene was also observed in the industrial KP_1.6366 strain (Fig. S8). In addition, we tested the capability of the pBECKP system for editing C-rich regions in the genome. Two C-rich spacers within the dhaK gene were assembled individually into the pBECKP-km plasmid. The plasmids were transformed individually into the KP_1.6366 strain. As shown in Fig. 5D and S9, various editing products with the conversions of Cs at different positions were obtained. These results demonstrated that the pBECKP system could efficiently convert C to T in a variety of K. pneumoniae strains.
The human BE3 base editor has a strong cytidine deamination capacity within the mutational spectra from positions 4 to 8 (termed the activity window) (16). Within the activity 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 the conversion efficiency was reduced drastically. Because the activity window of a cytidine base editor may not be identical in different species, we systematically examined the activity window and the sequence context preference of the pBECKP system in K. pneumoniae. Ten distinct spacers containing Cs at different positions were assembled into the pBECKP plasmid. The plasmids were transformed into the KP_1.6366 strain. As shown in Fig. 6, the editing efficiency of TC was higher than that of CC and AC. GC had the lowest editing efficiency, consistent with the sequence context preference of the mammalian base editor (16). The Cs from the TC motif at positions 3 to 8 were converted to Ts with efficiencies of almost 100%, whereas the editing efficiencies of the Cs at other positions were much lower, indicating that the activity window of the pBECKP system was from positions 3 to 8. Intriguingly, a few Cs at position 9 of spacer 8 and position 7 of spacer 10 were mutated to As but not Ts (Fig. 6). The editing by-product was also observed in the editing process of eukaryotic base editors (33–35).
FIG 6.
Determination of the activity window and sequence context preference of the pBECKP system in K. pneumoniae. The Cs with high editing efficiencies were marked with red squares. A few Cs at position 9 of spacer8 and position 7 of spacer10 were mutated to As but not Ts. These sites were colored green.
Dissection of drug resistance mechanisms using the two editing systems.
The quick dissemination of carbapenem-resistant K. pneumoniae has posed a severe threat to public health worldwide. The mobile genetic elements encoding carbapenemases dramatically accelerate the global expansion of carbapenem resistance. The acquirable carbapenemases 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 used both the pCasKP-pSGKP and pBECKP systems to verify the contribution of carbapenemases in carbapenem resistance.
First, we sought to delete the genes encoding carbapenemases and ESBLs individually in a hypermucoviscous carbapenem-resistant K. pneumoniae strain, KP_CRE23, using the pCasKP-pSGKP system. The KP_CRE23 strain harbored one carbapenemase gene, blaKPC-2, and two ESBL genes, blaSHV and blaCTX-M-65 (37). Because the KP_CRE23 strain is resistant to kanamycin, the kanamycin marker in both the pSGKP-km and the pBECKP-km plasmids was replaced with the spectinomycin marker, resulting in the pSGKP-spe and the pBECKP-spe plasmids. As shown in Fig. 7A, we obtained the desired chromosomal blaSHV deletion mutant with an efficiency of 4/12. However, in the case of the deletions of the plasmid-borne blaKPC-2 and blaCTX-M-65 genes using the same method, neither the desired gene deletion bands nor the wild-type bands were amplified by PCR in all the tested colonies (Fig. S10). A possible reason is that in the absence 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 reported that Cas9 nuclease-mediated DSB on a plasmid can be used for plasmid removal in Gram-negative bacteria (38).
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-borne blaKPC-2 gene (B) and Q136 of the plasmid-borne blaCTX-M-65 gene (C) were successfully mutated to stop codons with efficiencies of 8/8 and 2/8, respectively, by the pBECKP system. (D) blaKPC-2 was the key gene for carbapenem resistance in K. pneumoniae strain KP_CRE23.
Next, we attempted to use the base editor pBECKP-spe to inactivate the blaKPC-2 and blaCTX-M-65 genes, because the pBECKP-spe system executed editing by the introduction of a single-stranded DNA break instead of a DSB. As shown in Fig. 7B and C, the blaKPC-2 and blaCTX-M-65 genes were successfully mutated, resulting in the introduction of premature stop codons. The editing efficiencies were 8/8 and 2/8, respectively. We then examined the imipenem (a major carbapenem class drug) susceptibilities of the wild-type KP_CRE23 strain and three mutant strains using the inhibition zone and MIC assays. As shown in Fig. 7D, inactivation of the blaKPC-2 gene drastically increased the bacterial susceptibility to imipenem, whereas no significant drug susceptibility difference was observed when the blaSHV gene was deleted or the blaCTX-M-65 gene was inactivated. These results verified that the blaKPC-2 gene was the key factor that contributed to carbapenem resistance in the hypermucoviscous K. pneumoniae strain KP_CRE23. These approaches can be applied to a more complex system to dissect the drug resistance mechanisms of K. pneumoniae.
DISCUSSION
Klebsiella pneumoniae is an important industrial microorganism and human pathogen, but traditional genetic manipulation in K. pneumoniae is often time-consuming and laborious. Therefore, more efficient and simple genetic tools are highly desirable. In this study, by harnessing the powerful DNA-cleaving ability of the engineered CRISPR-Cas9 system and the strong recombination capacity of the lambda Red system, we have developed a convenient and efficient two-plasmid system, pCasKP-pSGKP, for iterative and scarless chromosomal gene deletion and insertion in K. pneumoniae. We first constructed a single-plasmid CRISPR-Cas9 system which could efficiently cleave the genomic DNA of K. pneumoniae. Due to the lack of the nonhomologous end-joining pathway, the DSB created by Cas9 nuclease on the chromosome was lethal to K. pneumoniae. Although the repair templates had been supplied by cloning them into the single CRISPR-Cas9 plasmid, no desirable deletion mutants were obtained, indicating 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 the lambda Red recombination system, which efficiently repaired the cleaved genomic DNA with the utilization of any type of repair template, including ssDNA.
To further simplify the editing process, we have developed a highly efficient cytidine base-editing system, pBECKP, by fusing a cytidine deaminase to the Cas9 nickase, enabling precise C→T conversions in both the chromosome and the plasmids. The activity window and the preference of the adjacent base of the editable sites were systematically investigated in K. pneumoniae. The pBECKP system could irreversibly inactivate genes by converting four codons (CAA, CAG, CGA, and TGG) into premature stop codons in a programmable manner. One major limitation of inactivating genes via the pBECKP system arises from the limited PAM sites that are adjacent to the aforementioned four codons. The pBECKP requires the presence of a nearby NGG site located 13 to 18 bp away from an editable CAA, CAG, CGA, or TGG codon. The recently evolved 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 a variety of clinically isolated K. pneumoniae strains. By using both editing systems, we verified the carbapenem resistance mechanism of the multidrug-resistant hypermucoviscous K. pneumoniae strain KP_CRE23. Because the KP_CRE23 strain was resistant to kanamycin, the kanamycin marker in both the pSGKP-km and pBECKP-km plasmids was replaced with the spectinomycin marker. Given that some K. pneumoniae isolates were insensitive to apramycin and/or kanamycin, the antibiotic resistances may limit the applications of the pCasKP-pSGKP system and the pBECKP system for genetic manipulations in multidrug-resistant K. pneumoniae strains. By testing the drug sensitivity of several clinically isolated K. pneumoniae strains, we detected that the majority of them were sensitive to hygromycin B. Thereby, we constructed a new plasmid, pCasKP-hph, which could serve as an alternative option for genetic manipulation in those apramycin-resistant K. pneumoniae strains. The antibiotic marker could also be replaced with 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. To obtain a high editing efficiency and reduce potential off-target effects, the spacers used in this study were designed using the sgRNAcas9 software (40). The sgRNAcas9 software could screen all the suitable spacer sequences in the target genes and evaluate their potential off-target sites throughout the K. pneumoniae genome.
Overall, we have engineered the two-plasmid system pCasKP-pSGKP for genome editing 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 wide variety of investigations, such as gene characterization, drug discovery, and metabolic engineering, in K. pneumoniae and relevant Enterobacteriaceae species.
MATERIALS AND METHODS
Plasmids, 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 primers used in this study were purchased from Genewiz (Suzhou, China) and are listed in Table S1. Escherichia coli DH5α and K. pneumoniae strains were grown in lysogeny broth (LB) medium (per liter, 5 g of yeast extract, 10 g of tryptone, 10 g of NaCl [pH 7.2 to ∼7.4]). Antibiotics were added at the following concentrations: 30 to 50 μg/ml apramycin, 100 μg/ml hygromycin B, 50 μg/ml kanamycin, and 50 to 100 μg/ml spectinomycin for both the E. coli and K. pneumoniae strains.
TABLE 1.
Plasmids used in this study
| Plasmid | Descriptiona | Reference or source |
|---|---|---|
| pCasSA | Plasmid carrying bacterial Cas9 nuclease gene (Kmr Cmr) | 41 |
| pKOBEG-apr | Thermosensitive plasmid, carries lambda Red genes (Aprr) | 23 |
| pOSCAR | Cloning plasmid (Sper) | 43 |
| pCasPA | Plasmid carrying sacB gene (Tetr) | 42 |
| pnCasSA-BEC | Plasmid carrying rAPOBEC1-XTEN-Cas9(D10A) gene (Kmr Cmr) | 32 |
| pET28a | Low-copy-no. plasmid (Kmr) | Lab stock |
| pMD19-mcherry | Plasmid carrying mcherry gene (Ampr) | Lab stock |
| pMD19-hyg | Plasmid carrying hph gene (Ampr Hygr) | 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 arm | 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 expressing sgRNA in K. pneumoniae (Kmr) | This study |
| pSGKP-spe | Plasmid expressing 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 arm | 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 arm | 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 |
| 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 |
Kmr, kanamycin resistance; Cmr, chloramphenicol resistance; Aprr, apramycin resistance; Sper, spectinomycin resistance; Tetr, tetracycline resistance; Ampr, ampicillin resistance; Hygr, hygromycin B resistance.
TABLE 2.
Bacterial strains used in this study
| Strain | Description or genotype | Reference |
|---|---|---|
| E. coli DH5α | F− Φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK−, mK+) phoA supE44 λ− thi-1 gyrA96 relA1 | Lab stock |
| K. pneumoniae | ||
| KP_1.6366 | Wild-type industrial K. pneumoniae strain | 44 |
| KP_1.6366 dhaF | KP_1.6366 ΔdhaF | This study |
| KP_1.6366 dhaK | KP_1.6366 ΔdhaK | This study |
| KP_1.6366 fosA | KP_1.6366 ΔfosA | This study |
| KP_1.6366 fosA::mcherry | KP_1.6366 ΔfosA::mcherry | This study |
| KP_1.6366 dhaF dhaK | KP_1.6366 ΔdhaF ΔdhaK | This study |
| KP_1.6366 fosA W92 to stop | KP_1.6366 fosA W92 mutation to stop codon | This study |
| KP_3744 | Wild-type clinically isolated K. pneumoniae strain | Lab stock |
| KP_3744 pyrF | KP_3744 ΔpyrF | This study |
| KP_3744 fepB | KP_3744 ΔfepB | This study |
| KP_3744 ramA | KP_3744 ΔramA | This study |
| KP_5573 | Wild-type clinically isolated K. pneumoniae strain | Lab stock |
| KP_5573 fosA | KP_5573 ΔfosA | This study |
| KP_5573 pyrF | KP_5573 ΔpyrF | This study |
| KP_5573 fepB | KP_5573 ΔfepB | This study |
| KP_5573 ramA | KP_5573 ΔramA | This study |
| KP_5573 fosA W92 to stop | KP_5573 fosA W92 mutation to stop codon | This study |
| KP_CRE23 | Wild-type clinically isolated K. pneumoniae strain with multidrug resistance and hypermucoviscosity | 37 |
| KP_CRE23 blaSHV | KP_CRE23 ΔblaSHV | This study |
| KP_CRE23 blaKPC-2 W164 to stop | KP_CRE23 blaKPC-2 W164 mutation to stop codon | This study |
| KP_CRE23 blaCTX-M-65 Q136 to stop | KP_CRE23 blaCTX-M-65 Q136 mutation to stop codon | This study |
Plasmid construction.
The temperature-sensitive pCasKP-apr plasmid was constructed using the following procedures. The rpsL promoter was PCR amplified from the genomic DNA of the K. pneumoniae strain KP_1.6366. The gene encoding the Cas9 nuclease was amplified from the pCasSA plasmid (41). The aforementioned two fragments along with the NdeI_linearized pKOBEG-apr plasmid (23) were assembled together using In-Fusion cloning, resulting in the final plasmid pCasKP-apr. The pCasKP-hph plasmid was constructed by replacing the apramycin resistance gene of the pCasKP-apr plasmid with the hygromycin B resistance gene.
The sgRNA expression cassette was synthesized commercially by Genewiz (Suzhou, China). The cassette contained the following three elements: the constitutive J23119 promoter, two BsaI restriction sites for the insertion of a 20-bp spacer, and the sgRNA scaffold. The sgRNA expression cassette was cloned into the EcoRV-digested pUC57 vector, yielding the pUC57-sgRNA plasmid. Then, the sacB gene amplified from the pCasPA plasmid (42) was inserted into the HindIII-digested pUC57-sgRNA plasmid via In-Fusion cloning, resulting in the final PSGKP-km plasmid. The pSGKP-spe plasmid was constructed by replacing the kanamycin resistance gene of the pSGKP-km plasmid with the spectinomycin resistance gene.
The pBECKP-km plasmid was constructed with the following procedure. The low-copy-number plasmid backbone containing pBR322_origin, the rop gene, and the kanamycin resistance marker were amplified from the pET28a plasmid. The sgRNA expression cassette and the sacB gene were amplified from 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) was inserted into the HindIII site of the aforementioned plasmid to form the final all-in-one pBECKP-km plasmid. The pBECKP-spe plasmid was constructed by replacing the kanamycin resistance gene of the pBECKP-km plasmid with the spectinomycin resistance gene.
Preparation of competent cells and electroporation.
For the K. pneumoniae wild-type strain, 1 ml overnight culture from a fresh single colony was diluted into 100 ml of LB broth and incubated at 37°C. When the optical density at 600 nm (OD600) of the cell culture reached 0.5 to 0.7, the culture was immediately chilled on ice for 20 min and then harvested by centrifugation at 7,200 × g for 5 min. The supernatant was discarded, and the cells were resuspended by pipetting gently with 15 ml of sterile ice-cold 10% glycerol. The centrifugation and resuspension steps were repeated twice. Finally, the cells were resuspended with 1 ml of ice-cold 10% glycerol. Fifty-microliter aliquots were frozen in liquid nitrogen and stored at −80°C.
For the pCasKP-harboring K. pneumoniae strain, 1 ml of overnight culture from a fresh single colony was diluted into 100 ml of LB broth containing 30 μg/ml apramycin and incubated at 30°C. When the cell density reached an OD600 of approximately 0.2, 1 ml of 20% l-arabinose was added for induction of the lambda Red recombineering operon of pCasKP. After induction at 30°C for 2 h, the culture was prepared as electrocompetent cells in a way similar to that of the wild-type K. pneumoniae.
For electroporation, 50 μl of electrocompetent cells was thawed on ice for several minutes. Then, the cells were mixed with no more than 5 μl plasmid or donor template. The mixture was transferred into a 2-mm electroporation cuvette (Bio-Rad) and electroporated at 2.5 kV, 200 Ω, and 25 μF. After being pulsed, the cells were recovered in 1 ml antibiotic-free LB broth and incubated at 30°C for 1.5 h before being plated onto LB agar plates supplemented with the required antibiotics. The plates were incubated at 30°C overnight.
Genome editing and base editing.
The detailed protocols for spacer cloning, genome editing, base editing, and plasmid curing in K. pneumoniae are provided in the supplemental material.
Antimicrobial susceptibility assay.
For the inhibition testing, a fresh K. pneumoniae suspension was adjusted to a 0.5 McFarland turbidity standard and then diluted 10 times with saline. The diluted bacterial suspension was evenly coated onto a Mueller-Hinton (MH) agar plate. The plate was dried for 5 min. A fosfomycin (50 μg/tablet; Oxoid) or imipenem (10 μg/tablet; Oxoid) tablet was placed in the center of the aforementioned MH plate. The plate was incubated at 35°C for 20 h to produce the inhibition zones.
For the MIC assay, the MICs of imipenem for the carbapenem-resistant KP_CRE23 strain and three mutant strains were determined using the 96-well broth microdilution method recommended by the Clinical and Laboratory Standards Institute (45). In brief, a fresh K. pneumoniae suspension was adjusted to a 0.5 McFarland turbidity standard and then diluted 10 times with saline. The 2-μl diluted solutions with 5.0 × 106 CFU bacterial cells were inoculated into 200 μl MH liquid medium containing serial 2-fold dilution concentrations of imipenem (0.5 to ∼64 μg/ml). The imipenem-free MH liquid medium was used as the control. After incubation at 35°C for 20 h, the MICs of complete growth inhibition were determined by visual inspection.
Data availability.
All the plasmids constructed in this study were validated by PCR, enzyme digestion, and DNA sequencing. Their sequences were submitted to the GenBank database under the accession 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 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).
Supplementary Material
ACKNOWLEDGMENTS
We thank Jian Hao from the Shanghai Advanced Research Institute, Chinese Academy 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 (grants 91753127 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) to Y.W.; and the Shanghai Sailing Program (grant 18YF1416500) to W.C.
Two patent applications have been submitted for the two-plasmid genome-editing system pCasKP-pSGKP and the single-plasmid base-editing system pBECKP.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01834-18.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
All the plasmids constructed in this study were validated by PCR, enzyme digestion, and DNA sequencing. Their sequences were submitted to the GenBank database under the accession 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 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).