CRISPR-Cas12a-Assisted Recombineering in Bacteria

• Published in: Applied and environmental microbiology 2017-09, Vol.83 (17)
• Main Authors: Yan, Mei-Yi, Yan, Hai-Qin, Ren, Gai-Xian, Zhao, Ju-Ping, Guo, Xiao-Peng, Sun, Yi-Cheng
• Format: Article
• Language: English
• Subjects: Antibiotic resistance; Antibiotics; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR-Cas Systems; E coli; Endonucleases - genetics; Endonucleases - metabolism; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Genetic Engineering; Genetics; Genome editing; Genomes; Genomics; Methods; Mutation; Mycobacterium smegmatis - enzymology; Mycobacterium smegmatis - genetics; Mycobacterium smegmatis - metabolism; Plasmids; Plasmids - genetics; Plasmids - metabolism; Recombinants; Recombination, Genetic; Yersinia pestis - enzymology; Yersinia pestis - genetics; Yersinia pestis - metabolism
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.00947-17

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 , , and 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. 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.