CRISPR-Cas12a-Assisted Recombineering in Bacteria

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria.IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.

Keywords: Cas12a; Mycobacterium smegmatis; Yersinia pestis; recombineering.

FIG 1

CRISPR-Cas12a-assisted genome editing in E. coli. (A) Schematic of CRISPR-Cas12a coupled with the λ Red system in E. coli. First, Cas12a and recombinase were expressed in bacteria. Then, the crRNA-expressing plasmid and ssDNA (or dsDNA) were transformed into the cell. When the crRNA targets the E.coli lacZ locus, wild-type E. coli dies or forms a blue colony, whereas the mutant forms a white colony on the X-Gal plate. (B) Schematic showing the crRNA and oligonucleotides used for editing of the E. coli lacZ locus. Cleavage sites are indicated by red arrows. The oligonucleotides lacZ.lag (59 nt) and lacZ.lead (59 nt, reverse and complementary sequence of lacZ.lag) were designed to mutate the PAM sequence and introduce a stop codon within the lacZ open reading frame. LacZ.del was designed to delete 1,399 bp from the lacZ gene. (C) The number and percentage of white colonies of transformants from the electroporation of the indicated pcrRNA plasmids and oligonucleotides (oligo) into E. coli MG1655 expressing recombinase and Cas12a. The transformation efficiency was defined as the total number of CFU generated per transformation. The transformants were plated on X-Gal plates for blue-white screening. The results are the averages of the results from at least two independent experiments, and the error bars depict the standard deviations.

FIG 2

CRISPR-Cas12a-assisted plasmid editing in Y. pestis. Generation of caf1R mutations in the pMT plasmid using CRISPR-Cas12a-assisted ssDNA oligonucleotide recombineering. The 59-nt recombinogenic oligonucleotides targeting the lagging strand or the leading strand of DNA replication were utilized for mutation. The transformation efficiency (A), the recombination efficiency (B), and the loss percentage of plasmid (C) are shown as the averages of the results from two independent experiments. ctrl, a control oligonucleotide with no homology to the genome of Y. pestis. Twenty colonies from each transformation were picked to test for plasmid loss and recombination by colony PCR and sequencing.

FIG 3

CRISPR-Cas12a-assisted single-stranded oligonucleotide recombineering in M. smegmatis. (A) Schematic showing the sequence of the gfp-targeting crRNA and oligonucleotides used for recombineering. This region corresponds to nucleotides 65 to 249 of the gfp ORF. The 60-nt recombinogenic oligonucleotides targeting the lagging strand (gfp1.lag and gfp2.lag) or targeting the leading strand (gfp1.lead) of DNA replication were utilized to disrupt the gfp gene by introducing changes in five consecutive base pairs (shown in brown) to generate two consecutive in-frame stop codons (lowercase and italic). The green and top-lined sequences represent the PAM. (B) Transformation and gfp recombination efficiency resulting from electroporation of the indicated pcrRNA plasmids and oligonucleotides into M. smegmatis expressing recombinase and Cas12a (FnCpf1). The transformation efficiency was defined as the total number of CFU generated per transformation, and the recombination efficiency was measured by determining the proportion of GFP-negative colonies. ATc (50 ng/ml) was added to the agar to induce Cas12a expression. Results are the averages of the results from at least two independent experiments, and the error bars depict the standard deviations.

FIG 4

Generation of subtle gene mutations using CRISPR-Cas12a-assisted ssDNA oligonucleotide recombineering in M. smegmatis. (A) Schematic showing the sequences of the point mutations generated in the gfp gene. Mutated sequences are shown in red. (B) Generation of point mutations described in panel A using CRISPR-Cas12a-assisted ssDNA oligonucleotide recombineering. (C) Schematic showing the sequences of 1-bp frameshifts generated in the gfp gene. Inserted sequences are shown in red, and the dashed line indicates the deleted sequence. (D) Generation of 1-bp insertions or deletions described in panel C using CRISPR-Cas12a-assisted ssDNA oligonucleotide recombineering. Transformation efficiency was defined as the total number of CFU generated per transformation, and recombination efficiency was measured by determining the proportion of GFP-negative colonies. Results are the averages of the results from at least two independent experiments, and the error bars depict the standard deviations.

FIG 5

Gene deletion and insertion using CRISPR-Cas12a-assisted single-stranded oligonucleotide recombineering in M. smegmatis. (A) Schematic of gene deletions and insertions generated in the gfp gene using CRISPR-Cas12a-assisted single-stranded oligonucleotide recombineering in M. smegmatis. (B) Generation of deletions and insertions shown in panel A using CRISPR-Cas12a-assisted single-stranded oligonucleotide recombineering in M. smegmatis. Introduction of a 5-, 10-, 20-, 418-, or 1,000-bp deletion or 5-, 10-, or 20-bp insertion in the gfp gene used CRISPR-Cas12a-assisted single-stranded oligonucleotide recombineering. The transformation efficiency was defined as the total number of CFU generated per transformation, and recombination efficiency was measured by determining the proportion of GFP-negative colonies. Results are the averages of the results from at least two independent experiments, and error bars depict standard deviations.

FIG 6

Double-stranded DNA recombineering assisted by CRISPR-Cas12a. Deletions of 2, 392, 1,000, or 4,000 bp were introduced into M. smegmatis chromosomal DNA using approximately 1-kb double-stranded DNA fragments with induced Cas12a. The gfp gene was replaced with a dsDNA PCR fragment containing the Hyg resistance gene or Ms5635–Ms5634 and its flanking region with induced Cas12a. Diagrams of the above gene deletions and replacements are shown in Fig. S5. Transformation efficiency was defined as the total number of CFU generated per transformation, and recombination efficiency was measured by determining the proportion of GFP-negative colonies. Results are the averages of the results from at least two independent experiments, and error bars depict standard deviations.