Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

Abstract

To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and "scar"-less.

FIG 1

Single-stranded oligonucleotide recombineering demonstration using the proposed three-plasmid system. (A) Disruption of chromosomal dbpA in E. coli. A 60-nucleotide recombinogenic oligonucleotide targeting the lagging strand of DNA replication (dbpA::STOP.lag) was utilized to disrupt dbpA by introducing six consecutive base pair changes and an AseI recognition sequence (lowercase), generating two consecutive in-frame stop codons (underlined). Sequence corresponding to the dbpA protospacer and PAM are shown in white. Rep corresponds to 36-bp repeats necessary for crRNA processing. (B) Electroporation and dbpA recombination efficiency data resulting from electroporation (pCRISPR::ctrl plus ctrl.60mer), CRISPR/Cas9 (pCRISPR::dbpA plus ctrl.60mer), and recombineering (pCRISPR::ctrl plus dbpA::STOP.lag) controls, as well as CRISPR/Cas9-coupled recombineering using leading or lagging strand oligonucleotides (pCRISPR::dbpA plus dbpA::STOP.lead or dbpA::STOP.lag). Electroporation efficiency is defined as the total number of CFU generated per microgram of plasmid DNA (pCRISPR::ctrl or pCRISPR::dbpA), and recombination efficiency was measured by determining the proportion of mutant colonies following PCR screening and AseI digestion. Recombination efficiency at the dbpA locus was not determined (ND) for electroporation of the nonrecombinogenic control oligonucleotide (ctrl.60mer). Results are averages of at least two independent experiments, and error bars depict standard deviations. (C) Colony PCR and AseI digestion screening of oligonucleotide-mediated dbpA gene disruption. A representative 13 colonies were used as the template in colony PCR with primers dbpA1.Fw and dbpA1.Rv, yielding a product of 1,002 bp. Successful oligonucleotide recombination generates products of 464 bp and 538 bp upon AseI digestion. Lane 1, marker; lanes 2, 4, 7, 8, and 13, negative, unmodified colonies; lanes 3, 5, 6, 9 to 12, and 14, positive, dbpA gene disruption colonies.

FIG 2

Double-stranded DNA gene replacement demonstration using the proposed three-plasmid system. (A) Replacement of 8-bp (H0 plus H0) and 818-bp (H1 plus H2) regions of dbpA with a 560-bp recombinogenic PCR product yielding lacZ′ (α). PCR primers possessing different Hn homology regions corresponding to 8-bp and 818-bp chromosomal deletions were utilized. Successful gene replacement replaces the dbpA PAM sequence and prevents Cas9 from generating a chromosomal DSB, which are both located between each set of homology regions. Genomic layout, primer binding sites, and expected PCR product sizes are shown for each dbpA gene replacement mutant, in addition to the wild-type strain. Genes corresponding to dbpA and lacZ′ are depicted to scale. “Rep” corresponds to 36-bp repeats necessary for crRNA processing. (B) Electroporation and dbpA recombination efficiency data resulting from electroporation (pCRISPR::ctrl plus control PCR product), CRISPR/Cas9 (pCRISPR::dbpA plus control PCR product), and recombineering (pCRISPR::ctrl plus 8-bp or 818-bp deletion cassette) controls, as well as an 8-bp or 818-bp chromosomal deletion using CRISPR/Cas9-coupled recombineering (pCRISPR::dbpA plus 8-bp or 818-bp deletion cassette). Electroporation efficiency is defined as the total number of CFU generated per microgram of plasmid DNA (pCRISPR::ctrl or pCRISPR::dbpA), and dbpA recombination efficiency was measured by determining the proportion of blue colonies. Recombination efficiency at the dbpA locus was not determined (ND) for electroporation of the nonrecombinogenic control PCR product. Results are averages of at least two independent experiments, and error bars depict standard deviations. (C) Colony PCR screening of dbpA gene replacement with lacZ′. Primers dbpA1.Fw and dbpA1.Rv were utilized in a colony PCR with one white and one blue colony of each gene replacement mutant type. Lane 1, marker; lane 2, wild-type E. coli colony; lanes 3 and 4, 8-bp gene replacement colonies; lanes 5 and 6, 818-bp gene replacement colonies; lanes 3 and 5, white (negative) colonies; lanes 4 and 6, blue (positive) colonies.

FIG 3

Challenging the proposed strategy of dsDNA gene replacement by varying chromosomal gene deletion size. (A) Replacement of various-size chromosomal regions with a recombinogenic PCR product encoding lacZ′ (α). PCR primers possessing different Hn homology regions corresponding to chromosomal deletions of various sizes (818 to 19,378 bp) were used to amplify a 560-bp lacZ′ product from pUC19. In each case, successful gene replacement replaces the dbpA PAM sequence and averts generation of a chromosomal DSB, which are both located between each set of homology regions. Genomic layout between the essential racR and insH genes (white) is shown for each gene replacement mutant. Chromosomal gene orientation is shown based on the dbpA coding sequence in the reverse (antisense) orientation. Primer binding sites and expected PCR product sizes are also depicted for each gene replacement mutant, in addition to the wild-type strain. All chromosomal genes and the lacZ′ PCR product are depicted to scale. (B) Electroporation and dbpA recombination efficiency data resulting from electroporation (pCRISPR::ctrl plus control PCR product), CRISPR/Cas9 (pCRISPR::dbpA plus control PCR product), and recombineering (pCRISPR::ctrl plus 818-bp or 2,428-bp PCR deletion cassette) controls, as well as six various-size chromosomal deletions using CRISPR/Cas9-coupled recombineering (pCRISPR::dbpA plus PCR deletion cassette). Electroporation efficiency is defined as the total number of CFU generated per microgram of plasmid DNA (pCRISPR::ctrl or pCRISPR::dbpA), and dbpA recombination efficiency was measured by determining the proportion of blue colonies. A recombination efficiency of 0% reflects an inability to identify blue mutant colonies on agar plates containing up to 400 transformants. Recombination efficiency at the dbpA locus was not determined (ND) for electroporation of the nonrecombinogenic control PCR product. Results shown are averages of at least two independent experiments, and error bars depict standard deviation. (C) Colony PCR screening of gene replacement mutants. Various PCR primers were used to ensure successful genomic organization by screening one white colony and one blue colony resulting from each gene replacement scheme. Lane 1, marker; lanes 2 and 3, 818-bp gene replacement colonies; lanes 4 and 5, 2,428-bp gene replacement colonies; lanes 6 and 7, 5,123-bp gene replacement colonies; lanes 8 and 9, 9,590-bp gene replacement colonies; lanes 10 and 11, 11,068-bp gene replacement colonies; lanes 12 and 13, 19,378-bp gene replacement colonies; lanes 2, 4, 6, 8, 10, and 12, white (negative) colonies; lanes 3, 5, 7, 9, 11, and 13, blue (positive) colonies. White colonies corresponding to lanes 4, 6, 8, 10, and 12 are expected to generate a PCR product that is too large to be amplified under the conditions employed.

FIG 4

Challenging the proposed strategy of dsDNA gene replacement by varying dsDNA insertion size. (A) Replacement of an 818-bp region of the dbpA gene with recombinogenic PCR products of various sizes yielding lacZ′ (α). Products were PCR amplified from plasmid pUC19 (560-bp, 1,264-bp, 1,756-bp, and 2,492-bp products) or pTH18cr (550-bp and 3,000-bp products) using PCR primers possessing H1 and H2 homology regions. Regions corresponding to the β-lactamase (Ap^r) coding sequence, ColE1 RNA (ColE1), and repA coding sequence (pSC101) are truncated in the 1,264-bp, 2,492-bp, and 3,000-bp products, respectively. In each case, successful gene replacement replaces the dbpA PAM sequence and avoids creation of a chromosomal DSB, which are both located between the H1 and H2 homology regions. The expected genomic layout at the dbpA locus is shown for each gene replacement mutant. Primer binding sites and expected PCR product sizes are also depicted for each dbpA gene replacement mutant, in addition to the wild-type strain. Genes and plasmid regions corresponding to dbpA, lacZ′, Ap^r, Cm^r, ColE1, and pSC101 sequences are depicted to scale. Note that size of the lacZ′ coding sequence differs between plasmids pUC19 (324 bp) and pTH18cr (237 bp). (B) Electroporation and dbpA recombination efficiency data resulting from electroporation (pCRISPR::ctrl plus control PCR product), CRISPR/Cas9 (pCRISPR::dbpA plus control PCR product), and recombineering (pCRISPR::ctrl plus 550-bp, 560-bp, or 1,264-bp PCR insertion cassette) controls, as well as six various-size chromosomal deletions using CRISPR/Cas9-coupled recombineering (pCRISPR::dbpA plus PCR insertion cassette). Electroporation efficiency is defined as the total number of CFU generated per microgram of plasmid DNA (pCRISPR::ctrl or pCRISPR::dbpA), and dbpA recombination efficiency was measured by determining the proportion of blue colonies. A recombination efficiency of 0% reflects an inability to identify blue mutant colonies on agar plates containing up to 400 transformants. Recombination efficiency at the dbpA locus was not determined (ND) for electroporation of the nonrecombinogenic control PCR product. Results shown are averages of at least two independent experiments, and error bars depict standard deviations. (C) Colony PCR screening of dbpA gene replacement mutants. Primers dbpA1.Fw and dbpA1.Rv were utilized in a colony PCR with one blue colony resulting from each gene replacement scheme. Lane 1, marker; lane 2, wild-type E. coli colony; lane 3, 550-bp insertional colony; lane 4, 560-bp insertional colony; lane 5, 1,264-bp insertional colony; lane 6, 1,756-bp insertional colony; lane 7, 2,492-bp insertional colony; lane 8, 3,000-bp insertional colony.

FIG 5

Comparison of the proposed CRISPR/Cas9-coupled recombineering methodology with previous strategies of E. coli chromosomal gene replacement in the absence of CRISPR/Cas9 (9, 10). All technical steps involved in each protocol to obtain the target markerless gene replacement mutant strain are depicted. Previous methods result in a mutant strain possessing an FRT scar site, whereas the mutant derived from the protocol proposed in this study generates a scarless mutant. AB^R, antibiotic resistance marker.