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Multiplex Generation, Tracking, and Functional Screening of Substitution Mutants Using a CRISPR/Retron System

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journal contribution
posted on 04.05.2020, 18:40 by Hyeonseob Lim, Soyeong Jun, Minjeong Park, Junghak Lim, Jaehwan Jeong, Ji Hyun Lee, Duhee Bang
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.