We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/retron system for multiplexed generation of substitution mutations by coutilization of a retron system that continuously expresses donor DNA and a CRISPR/Cas9 cassette that induces cleavage at target genomic loci. Our system efficiently introduces substitution mutation in the Escherichia coli genome in a high-throughput manner. These substitution mutations can be tracked by analysis of retron plasmid sequences without laborious amplification of individual edited loci. We demonstrated that our CRISPR/retron system can introduce thousands of mutations in a single experiment and be used for screening phenotypes related to chemical responses or fitness changes. We expect that our system could facilitate genome-scale substitution screenings.
Bibliographical notePublisher Copyright:
© 2020 American Chemical Society.
All Science Journal Classification (ASJC) codes
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)