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.

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…In contrast, both SpCas9‐ and nSpCas9‐expressing plasmids are barely transformable. Moreover, Cas12a nucleases were proved to be practical in gene editing in other bacteria . In addition, since near half bacteria harbor intrinsic CRISPR‐Cas systems, reprogramming their systems to target themselves would eliminate the toxicity issue from exogenous Cas nuclease expression and greatly simplify the editing system.…”

“…Currently, two auxiliary recombination systems, λ Red and RecE/T, have been widely adopted in CRISPR‐based genome editing for many genera, such as Acinetobacter , Escherichia , Enterobacter , Klebsiella , Lactobacillus , Pseudomonas , Tatumella , and Yersinis (Table S1, Supporting Information). These systems allow the DSB repair using templates as linear dsDNA, such as PCR products, or synthetic single‐strand oligonucleotides, which dramatically reduces the labor and time consumption in plasmid construction.…”

“…Similarly, van Kessel and Hatfull had characterized a rare RecE/T homolog from a mycobacteriophage (Che9c) in Mycobacterium tuberculosis , consisting of two proteins, Che9c gp60, and gp61. Yan et al integrated Che9c gp60 and gp61 with FnCas12a, enabling precise and efficient genome editing in M. smegmatis .…”

“…Second, targeting of the ss oligos to a lagging strand in a replication fork could improve the efficiency of the conventional SSAP‐mediated homologous recombination . This approach was also confirmed to be practical in repairing Cas nuclease‐delivered DSB . Third, avoiding the intrinsic mismatch repair (MMR) system, MutS, could substantially benefit to recombination efficiency in creating site‐direct mutagenesis, as MMR reverts successful edits back to the wild type.…”

A highly efficient in vivo plasmid editing tool based on CRISPR-Cas12a and phage λ Red recombineering

“…In addition, multimeric plasmids or mixtures of recombinant and parental plasmids are formed when plasmids are modified using recombineering, thereby limiting its application, especially in the case of multicopy plasmids (Yu et al, 2000;Thomason et al, 2007). The CRISPR-Cas endonuclease system, which generates programmed double-stranded DNA (dsDNA) cleavage, is an effective genome editing tool (Cong et al, 2013;Yan et al, 2017;Javed et al, 2018). Here, we combined CRISPR-Cas12a and recombineering approaches to establish an efficient plasmid editing system ( Fig.…”