CRISPR/Cas9-Mediated Site-Directed Mutagenesis of Genes in Klebsiella pneumoniae

Summary

This study describes a CRISPR/Cas9-based protocol for constructing precise point mutations in Klebsiella pneumoniae to investigate antimicrobial resistance mechanisms. It exploits the strict PAM requirement of Cas9 and codon degeneracy using a repair template that simultaneously introduces the desired mutation and disrupts the PAM, preventing re-cleavage and enhancing screening specificity.

Abstract

Constructing target-gene mutants with a common genetic background is crucial for elucidating gene function in antimicrobial resistance (AMR) research. Taking advantage of the single-guide RNA (sgRNA) and protospacer adjacent motif (PAM) sequence (3’-NGG) specificity of the Cas9 protein in the CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system and codon degeneracy, the authors design a repair template that incorporates the desired point mutation while excluding the PAM sequence disrupted by a synonymous substitution, thereby preventing re-cleavage by CRISPR/Cas9. This method employs two available plasmids, pCasKP-apr and pSGKP-spe, offering straightforward operation and high screening specificity. As a result, the approach enables efficient generation of genetically defined mutant strains of Klebsiella pneumoniae (K. pneumoniae) and is readily adaptable to routine laboratory settings. Furthermore, the protocol minimizes off-target editing, shortens experimental timelines, reduces screening workload, and provides a reliable platform for investigating resistance mechanisms, validating candidate genes, and supporting functional genomics studies in clinically relevant bacterial pathogens.

Introduction

Drug-resistant K. pneumoniae poses a growing threat to public health, as carbapenem-resistant and hypervirulent lineages continue to drive high rates of treatment failure and mortality^1,2,3. A deeper understanding of the molecular determinants that drive antimicrobial resistance is urgently needed, as it can directly inform the development of novel antibacterial agents and help improve the management and prognosis of patients with severe infections⁴. Establishing a direct link between a gene and resistance requires the construction of isogenic mutants that carry only the mutation of interest, removing the influence of unrelated background variation. Generating such precise mutants in K. pneumoniae has been challenging because widely accessible, reproducible genetic tools for point mutagenesis remain limited.

Conventional allelic-exchange methods that rely on suicide vectors and counterselection markers are often labor-intensive, require cloning of long homology arms, and produce low recombination frequencies in many clinical K. pneumoniae isolates^5,6,7. The application of CRISPR/Cas9 genome editing has accelerated bacterial genetics by supplying a sequence-specific counterselection that strongly favors the recovery of edited cells^8,9. The majority of published CRISPR/Cas9 approaches for Enterobacteriaceae, however, have been designed for gene inactivation or deletion and typically still demand assembly of new plasmids for each target, long repair templates, and multiple screening rounds^10,11,12. Because many antimicrobial resistance phenotypes are caused by single-nucleotide substitutions in specific resistance genes, a dedicated point-mutation protocol is clearly needed. Here, the authors outline a streamlined procedure for installing precise, markerless point mutations in K. pneumoniae that relies exclusively on two accessible plasmids, pCasKP-apr and pSGKP-spe, originally developed for efficient editing in this species⁸. Notably, the two-plasmid system consisting of pCasKP-apr and pSGKP-spe has been successfully applied for gene inactivation and large-fragment deletion in K. pneumonia.

The method takes advantage of the strict PAM (3′-NGG) requirement of Cas9 protein, and the degeneracy of the genetic code—a short repair template is designed to carry the desired base change while also creating a synonymously mutated PAM sequence that is no longer recognized by Cas9. This dual modification is intended to prevent re-cleavage of the edited chromosome and to improve screening stringency. Under the conditions tested, this approach reduced the number of colonies that needed to be examined before the correct edit was found. By consolidating several steps, the protocol aims to make precise genome editing in K. pneumoniae more accessible to laboratories without specialized equipment, thereby facilitating genotype–phenotype analyses and supporting further study of AMR mechanisms.

Protocol

1. Biosafety statement

NOTE: All work with K. pneumoniae was performed in a biosafety level 2 (BSL-2) facility in strict accordance with the institutional biosafety guidelines and approved protocols. The experiments shown in this protocol were conducted using a carbapenem-resistant clinical isolate of K. pneumoniae (strain ID CRKP288). This strain was handled exclusively under BSL-2 containment, and appropriate personal protective equipment was worn at all times. To concretely illustrate the workflow, the following steps describe the introduction of the missense mutation c.227A>T (p.Asp76Val) into the gene ftsI, which encodes penicillin-binding protein 3 (PBP3) in K. pneumoniae¹³. The procedure can be applied to any chromosomal locus by redesigning the sgRNA and repair template accordingly.

2. Chemical transformation of pCasKP-apr into the target K. pneumoniae strain

1. Prepare chemically competent cells.
   1. Streak the target K. pneumoniae isolate from a −80 °C glycerol stock onto an Luria broth (LB) agar plate. Incubate at 37 °C overnight. Inoculate a single colony into 5 mL of LB broth. Incubate at 37 °C with shaking at 180 rpm overnight.
   2. Dilute the overnight culture 1:100 into 50 mL of fresh LB broth in a 250 mL flask. Incubate at 37 °C with shaking until the optical density at 600 nm (OD₆₀₀) reaches 0.5–0.6. Chill the culture on ice for 15 min.
   3. Transfer the culture to a pre-chilled, sterile 50 mL centrifuge tube. Harvest the cells by centrifugation at 4000 × g for 10 min at 4 °C. Discard the supernatant.
   4. Gently resuspend the cell pellet in 16 mL of Ultra-Competent Cell Preps Kit BT Buffer A (see Table of Materials). Incubate on ice for 30 min.
   5. Centrifuge at 4,000 × g for 10 min at 4 °C, discard the supernatant, and gently resuspend the cells in 4 mL of BT Buffer B.
   6. Divide the suspension into 100 µL aliquots in pre-chilled sterile 1.5 mL microcentrifuge tubes. Snap-freeze the aliquots in liquid nitrogen and store at −80 °C.
2. Chemical transformation
   1. Inoculate the E. coli strain carrying pCasKP-apr into 10 mL of fresh LB broth containing apramycin (50 µg/mL) and incubate overnight at 30 °C with shaking at 180 rpm.
   2. Extract the plasmid using a plasmid extraction kit and elute in 100 µL of nuclease-free water. Store the purified pCasKP-apr plasmid at −20 °C.
   3. Thaw one aliquot of the competent K. pneumoniae cells on ice. Add 10 µL of pCasKP-apr plasmid DNA to the cells. Mix by gently flicking the tube 4–5 times, and incubate the mixture on ice for 30 min.
   4. Heat-shock the cells by placing the tube in a 42 °C water bath for 90 s. Ensure that the bottom half of the tube is submerged.
   5. Immediately return the tube to ice and incubate for 2 min. Add 900 µL of pre-warmed SOC medium (without antibiotic) to the tube. Incubate at 30 °C for 2 h with gentle shaking at 180 rpm.
      ​NOTE: The incubation temperature of 30 °C is essential because the pCasKP-apr plasmid carries a temperature-sensitive replicon. Higher temperatures prevent stable plasmid replication.
   6. Concentrate the culture by centrifugation at 4,000 × g for 2 min, discard most of the supernatant, resuspend the pellet in approximately 100 µL of residual medium, and spread onto an LB agar plate containing apramycin (50 µg/mL). Incubate the plates at 30 °C for 24–36 h.
   7. Select 3–5 individual colonies and verify the presence of pCasKP-apr by PCR (Polymerase chain reaction) using the primers listed in Table 1. Once the plasmid is confirmed, prepare a glycerol stock of the verified K. pneumoniae (pCasKP-apr) strain and store it at −80 °C.

3. Design of the sgRNA and repair template

1. Scan the sequence for a PAM (5′-NGG-3′) in the vicinity of the intended mutation site using an online CRISPR design tool, Benchling (https://www.benchling.com/crispr). Select a 20-NT protospacer sequence immediately upstream of the PAM.
   ​NOTE: For the exemplary ftsI c.227A>T (p.Asp76Val) mutation, use the protospacer 5′-CAGCGGACGTCCGGAGCGAT-3′ with the adjacent PAM 5′-CGG-3′.
2. Digestion and purification of the pSGKP-spe vector
   1. Inoculate the E. coli strain carrying pSGKP-spe into 10 mL of fresh LB broth supplemented with spectinomycin (30 µg/mL). Incubate overnight at 37 °C with shaking at 180 rpm.
   2. Extract the plasmid using a plasmid extraction kit and elute in 100 µL of nuclease-free water. Store the purified pSGKP-spe plasmid at −20 °C.
   3. Set up a restriction digestion to generate linear pSGKP-spe with cohesive ends. Prepare the following reaction in a sterile microcentrifuge tube (the reaction mixture is detailed in Table 2). Incubate at 37 °C for 1 h.
   4. Purify the linearized vector using a DNA purification kit to remove the enzyme and buffer. Elute in 30–50 µL of nuclease-free water. Store the linear pSGKP-spe at −20 °C until the ligation step.
   5. Design and anneal the sgRNA oligonucleotides. Append the overhang 5′-TAGT to the 5′ end of the protospacer to generate the forward oligonucleotide, and append the overhang 5′-AAAC to the 5′ end of its reverse complement to generate the reverse oligonucleotide.
   6. Phosphorylate the sgRNA oligonucleotides at 37 °C for 30 min in a sterile microcentrifuge tube (the reaction mixture is detailed in Table 3). Incubate the reaction solution at 95 °C for 5 min, then slowly cool to room temperature.
      NOTE: For the ftsI c.227A>T example, the underlined sequences denote the sgRNA oligonucleotides: ftsI forward oligonucleotide: 5′-TAGTCAGCGGACGTCCGGAGCGAT-3′;
      ftsI reverse oligonucleotide: 3′- GTCGCCTGCAGGCCTCGCTACAAA-5′.
   7. Ligate the annealed sgRNA insert into the linearized pSGKP-spe vector by incubating the reaction at 16 °C overnight in a sterile microcentrifuge tube (the reaction mixture is detailed in Table 4).
   8. Prepare chemically competent E. coli DH5α cells with a competent cell preparation kit (Table of Materials), transform them with the ligation mixture by heat shock (42 °C, 45 s), and obtain colonies carrying the pSGKP-spe-sgRNA plasmid.
   9. Design a repair template of 200–300 NT that carries the desired nucleotide substitution and simultaneously introduces a synonymous mutation that disrupts the PAM sequence without altering the encoded amino acid.
   10. Using Snapgene (Version 4.2.4)¹⁴ design primers to PCR-amplify both the repair template and the pSGKP-spe-sgRNA vector. Clone the repair template into the multiple cloning sites of the vector via the In-Fusion assembly method.
   11. Designate the pSGKP-spe-sgRNA-RT (repair template) plasmid, which carries both the sgRNA cassette and the repair template donor sequence.
       NOTE: For the exemplary ftsI c.227A>T (p.Asp76Val) point mutation, the primers are provided in Table 1. A schematic diagram of the pSGKP-spe-sgRNA-RT plasmid is shown in Figure 1. Transform the constructed pSGKP-spe-sgRNA-RT plasmid into chemically competent E. coli DH5α following the heat shock procedure described in step 2.2.

4. Preparation of electrocompetent K. pneumoniae (pCasKP-apr) cells and electroporation of pSGKP-spe-sgRNA-RT plasmid

1. Streak the verified K. pneumoniae (pCasKP-apr) strain (from step 2.2.6) from a −80 °C glycerol stock onto an LB agar plate containing apramycin (50 µg/mL). Incubate at 30 °C for 24–36 h.
2. Inoculate a single colony into 10 mL of fresh LB broth containing apramycin (50 µg/mL) and 0.2% (w/v) L-arabinose. Incubate at 30 °C with shaking until the OD₆₀₀ reaches 0.4–0.5.
   NOTE: The presence of L-arabinose induces expression of the λ-Red recombination system from the pCasKP-apr plasmid, which is required for efficient homologous recombination in the subsequent editing step.
3. Chill the culture on ice for 20 min. Harvest the cells by centrifugation at 4,000 × g for 10 min at 4 °C. Discard the supernatant.
4. Wash the cell pellet twice with 10 mL of ice-cold 10% (v/v) glycerol, centrifuging at 4,000 × g for 10 min at 4 °C after each wash. Resuspend the final pellet in 1 ml of ice-cold 10% glycerol.
5. Divide the suspension into 100 µL aliquots in pre-chilled sterile 1.5 mL microcentrifuge tubes. Use the aliquots immediately for electroporation or snap-freeze in liquid nitrogen and store at −80 °C.
   NOTE: Electrocompetent K. pneumoniae (pCasKP-apr) cells can be stored at −80 °C for up to 3 months. Thaw on ice immediately before use.
6. Thaw one aliquot of the electrocompetent cells on ice. Add 10 µL of the pSGKP-spe-sgRNA-RT plasmid (prepared in step 3.2.7) and gently flick the tube to mix. Transfer the mixture to a pre-chilled 0.2 cm Gene Pulsr cuvette.
7. Electroporate using the following settings: 2.2 kV, 25 µF capacitance, and 200 Ω resistance. Immediately add 1 mL of pre-warmed (30 °C) SOC medium containing 0.2% (w/v) L-arabinose to the cuvette, gently resuspend the cells, and transfer the suspension to a sterile culture tube. Incubate the tube at 30 °C for 4 h with gentle shaking (150–180 rpm).
8. Spread 100 µL of the culture onto separate LB agar plates supplemented with apramycin (50 µg/mL) and spectinomycin (30 µg/mL). Incubate the plates at 30 °C for 24–48 h until visible colonies appear.
9. Pick 5–10 individual colonies and screen for the desired point mutation by PCR amplification of the target locus followed by Sanger sequencing (Table 2).
   NOTE: The number of colonies may be substantially lower than in a standard transformation because Cas9-mediated double-strand breaks impose strong negative selection against unedited cells.

5. Curing of the pCasKP-apr and pSGKP-spe plasmids from the confirmed mutant

1. Inoculate the confirmed colony into 5 mL of antibiotic-free LB broth. Incubate the culture at 37 °C for 12 h with shaking at 180 rpm.
   NOTE: Incubation at 37 °C in the absence of antibiotic selection pressure promotes the loss of the temperature-sensitive pCasKP-apr replicon.
2. After the 12 h incubation, streak a fraction of the culture (e.g., a 10 µL loopful) directly onto an LB agar plate containing 5% (w/v) sucrose. Incubate the plate at 37 °C overnight.
   NOTE: The preparation procedure of 5% sucrose LB agar plates: Weigh 10 g of sucrose and dissolve it completely in 20 mL of sterile distilled water to obtain a 50% (w/v) stock solution. Sterilize the solution by filtration through a 0.22 µm sterile syringe filter (see Table of Materials); add the sterile sucrose stock to autoclaved LB agar medium at a 1:10 ratio to achieve a final sucrose concentration of 5% (w/v), mix gently, and pour the plates. The pSGKP-spe plasmid carries the sacB gene, conferring sucrose sensitivity. Only cells that have lost this plasmid will form colonies on the sucrose-containing medium. A sucrose concentration of 5% is typically sufficient for effective counterselection in K. pneumoniae.
3. Screen the putative cured clones for the loss of both plasmids. First, streak each clone in parallel onto three plates: (i) an LB agar plate without antibiotics, (ii) an LB agar plate containing apramycin (50 µg/mL), and (iii) an LB agar plate containing spectinomycin (30 µg/mL). Incubate at 37 °C overnight.
4. Identify clones that grow only on the antibiotic-free plate. Confirm the absence of both plasmids by colony PCR using specific primers (see Table 1) that target the Cas9 gene (for pCasKP-apr) and the gRNA scaffold (for pSGKP-spe). The absence of PCR amplification confirms successful plasmid clearance.
5. Prepare a glycerol stock of the verified, plasmid-cured mutant strain and store it at −80 °C.

Results

The overall goal of this protocol is to generate a markerless, defined point mutation in the K. pneumoniae chromosome using a two-plasmid CRISPR/Cas9 system. To illustrate the outcomes, the authors present the results for a representative target: introducing the c.227A>T (p.Asp76Val) missense mutation into the ftsI.

Construction of the pSGKP-spe-sgRNA-RT

The successful construction of the pSGKP-spe-sgRNA-RT plasmid, which simult...

Discussion

The protocol presented here enables the efficient introduction of defined, markerless point mutations into the K. pneumoniae chromosome by combining CRISPR/Cas9 counterselection with a two-plasmid system. Several steps require particular attention to achieve the high editing efficiencies reported. The most critical design element is the repair template, which must simultaneously introduce the desired nucleotide change and create a synonymous mutation that disrupts the PAM without altering the encoded amino acid....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm Sterile syringe filter	Biosharp	BS-PES13-22-S	Filter-sterilize the sucrose stock solution
2 × Rapid Taq Master Mix	Vazyme	P223-01	Fast PCR; Genotyping; Colony PCR
Gene Pulser/MicroPulser Electroporation Cuvettes, 0.2 cm gap	BIO-RAD	#1652086	For reproducible transformation of bacteria, yeast, and mammalian cells, including primary and stem cells
In-Fusion Snap Assembly Master Mix	TaKaRa	638948	Insert one or more PCR fragments into any desired position of any vector according to the design specifications.
LB Broth	Hopebio	HB0128	For bacterial enrichment culture.
LB Nutrient Agar	Hopebio	HB0129	For the cultivation of bacteria in fermentation engineering, genetic engineering, and molecular biology.
MinElute PCR Purification Kit for PCR Cleanup	QIAGEN	28004	The MinElute PCR Purification procedure removes primers, nucleotides, enzymes, mineral oil, salts, and other impurities from DNA samples
pCasKP-apr	Addgene	Plasmid #117231	Bacterial Expression, CRISPR, Synthetic Biology
PrimeSTAR HS (Premix)	TaKaRa	R040Q	A 2× concentration Premix product for PCR applications, containing DNA Polymerase, Buffer, and dNTP Mixture.
pSGKP-spe	Addgene	Plasmid #117234	Bacterial Expression, CRISPR, Synthetic Biology
QIAGEN Plasmid Mini Kit	QIAGEN	12123	For purification of up to 10 mg transfection-grade plasmid or cosmid DNA
SOC Medium	Hopebio	HBDC002	For the cultivation of genetically engineered Escherichia coli.
SpeedyCut BsaI	Sangon Biotech-BBI	#B600769	A fast enzyme that can rapidly cut DNA within 5–15 min.
Sucrose	Sinopharm Chemical Reagent Co., Ltd.	10021418	Preparation of 5% (w/v) sucrose LB agar plates
T4 DNA Ligase	TaKaRa	2011A	Catalyzes the formation of a phosphodiester bond between the 5′-P end and the 3′-OH end of adjacent DNA strands
T4 Polynucleotide Kinase	TaKaRa	2021S	Labeling of the 5′ ends of DNA and RNA, 5′ end phosphorylation of synthetic DNA linkers.
Ultra-Competent Cell Preps Kit	Sangon Biotech	B529303	This kit uses a modified rubidium chloride method to prepare high-efficiency competent Escherichia coli cells