CRISPR-Cas9(D10A) Nickase-Assisted Genome Editing in Lactobacillus casei.

CRISPR-Cas9(D10A) Nickase-Assisted Genome Editing in Lactobacillus casei. Appl Environ Microbiol. 2017 Sep 01;: Authors: Song X, Huang H, Xiong Z, Ai L, Yang S Abstract Lactobacillus casei has drawn increasing attention as health-promoting probiotics, while effective genetic manipulation tools are often not available, e.g. the single gene knockout in L. casei still depends on the classic homologous recombination-dependent double crossover strategy that is quite labor intensive and time-requiring. In the present study, a rapid and precise genome editing plasmid pLCNICK was established for L. casei genome engineering based on CRISPR-Cas9(D10A) In addition to the P23-Cas9(D10A) and Pldh-sgRNA (single guide RNA) expression cassettes, pLCNICK includes the homologous arms of the target gene as repair templates. The ability and efficiency of chromosomal engineering using pLCNICK were evaluated by in-frame deletions of four independent genes and chromosomal insertion of an enhanced green fluorescent protein (eGFP) expression cassette at the LC2W_1628 locus. The efficiencies associated with in-frame deletions and chromosomal insertion is 25-62%. The pLCNICK has been proved to be an effective, rapid and precise tool for genome editing in L. casei and its potential application in other members of Lactic acid bacteria (LAB) was also discussed in this study.Importance The lack of efficient genetic tools has limited the investigation and biotechno...
Source: Applied and Environmental Microbiology - Category: Microbiology Authors: Tags: Appl Environ Microbiol Source Type: research