Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System

• Published in: Applied and Environmental Microbiology Vol. 81; no. 7; pp. 2506 - 2514
• Main Authors: Jiang, Yu, Chen, Biao, Duan, Chunlan, Sun, Bingbing, Yang, Junjie, Yang, Sheng
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.04.2015
• Subjects: Chromosomes; CRISPR-Cas Systems; E coli; Enterobacteriaceae; Enterobacteriaceae - enzymology; Enterobacteriaceae - genetics; Escherichia coli; Genes, Bacterial; Genetics; Genetics, Microbial - methods; Genome, Bacterial; Genomics; Gram-negative bacteria; Methods; Microbiology; Molecular Biology - methods; Recombination, Genetic; Streptococcus pyogenes; Streptococcus pyogenes - enzymology; Streptococcus pyogenes - genetics
• ISSN: 0099-2240; 1098-5336; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.04023-14

An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea , another species of the Enterobacteriaceae , with highest efficiency of 100%.