Rapid, Efficient, and Cost-Effective Gene Editing of Enterococcus faecium with CRISPR-Cas12a.

ABSTRACT: Considered a serious threat by the Centers for Disease Control and Prevention, multidrug-resistant Enterococcus faecium is an increasing cause of hospital-acquired infection. Here, we provide details on a single-plasmid CRISPR-Cas12a system for generating clean deletions and insertions. Single manipulations were carried out in under 2 weeks, with successful deletions/insertions present in >80% of the clones tested. Using this method, we generated three individual clean deletion mutations in the acpH, treA, and lacL genes and inserted codon-optimized unaG, enabling green fluorescent protein (GFP)-like fluorescence under the control of the trehalase operon. The use of in vivo recombination for plasmid construction kept costs to a minimum. IMPORTANCE

SUBMITTER: Chua MJ 

PROVIDER: S-EPMC8809335 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature