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

• Published in: ACS synthetic biology Vol. 9; no. 5; pp. 1003 - 1009
• Main Authors: Lim, Hyeonseob, Jun, Soyeong, Park, Minjeong, Lim, Junghak, Jeong, Jaehwan, Lee, Ji Hyun, Bang, Duhee
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.05.2020
• Subjects: Clustered Regularly Interspaced Short Palindromic Repeats - genetics; Escherichia coli - genetics; Gene Editing - methods; Gene Library; Genome, Bacterial; Mutation; Plasmids - genetics; Plasmids - metabolism; RNA, Guide, CRISPR-Cas Systems - metabolism; CRISPR; genome editing; recombineering; screening; multiplexing; retron
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.0c00002

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.