CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli.

ABSTRACT: Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly

SUBMITTER: Choudhury A 

PROVIDER: S-EPMC7073797 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature