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 language | English |
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Pages (from-to) | 1003-1009 |
Number of pages | 7 |
Journal | ACS Synthetic Biology |
Volume | 9 |
Issue number | 5 |
DOIs | |
Publication status | Published - 2020 May 15 |
Bibliographical note
Publisher Copyright:© 2020 American Chemical Society.
All Science Journal Classification (ASJC) codes
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)