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 noteFunding Information:
This work was supported by (i) the Midcareer Researcher Program (NRF-2018R1A2A1A05079172 to D.B., H.L., S.J.), (ii) the Bio & Medical Technology Development Program (NRF-2016M3A9B6948494 to D.B., H.L., S.J.), (iii) the Bio & Medical Technology Development Program (NRF-2018M3A9H3024850 to D.B., H.L., S.J.), and (iv) Basic Science Research Program (NRF-2018R1A2B2001322 to J.H.L., S.J.) of the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Planning, and (v) the Korea Health Technology R&D Project (HI18C2282 to J.H.L., D.B., H.L.) through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare.
© 2020 American Chemical Society.
All Science Journal Classification (ASJC) codes
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)