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 journalArticlepeer-review

3 Citations (Scopus)

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

Bibliographical note

Funding 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.

Publisher Copyright:
© 2020 American Chemical Society.

All Science Journal Classification (ASJC) codes

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

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