Multiplex generation, tracking, and functional screening of substitution mutants using a crispr/retron system

Hyeonseob Lim, Soyeong Jun, Minjeong Park, Junghak Lim, Jaehwan Jeong, Ji Hyun Lee, Duhee Bang

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)1003-1009
Number of pages7
JournalACS Synthetic Biology
Volume9
Issue number5
DOIs
Publication statusPublished - 2020 May 15

All Science Journal Classification (ASJC) codes

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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