Abstract
ABSTRACTClustered 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.
Highlights
Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms
Our results show that CRISPRCas12a-assisted recombineering can rapidly and efficiently generate point mutations, deletions, and insertions in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis
We described the development of a system that couples CRISPR-Cas12a genome editing with recombineering to allow efficient genetic manipulation in E. coli, Y. pestis, and M. smegmatis
Summary
Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. 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 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. Antibiotic resistance genes must be cured by a technique involving a site-specific recombinase or resolvase [17, 19,20,21] These manipulations require multiple steps and create a chromosomal “scar” (e.g., at the LoxP recognition site). CRISPR-Cas9-assisted genome editing has not yet been applied to this group of microorganisms
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