Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Efficient CRISPR-Cas12f1-Mediated Multiplex Bacterial Genome Editing via Low-Temperature Recovery

  • Se Ra Lim (Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University) ;
  • Hyun Ju Kim (Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University) ;
  • Sang Jun Lee (Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University)
  • Received : 2024.03.18
  • Accepted : 2024.05.29
  • Published : 2024.07.28
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Abstract

CRISPR-Cas system is being used as a powerful genome editing tool with developments focused on enhancing its efficiency and accuracy. Recently, the miniature CRISPR-Cas12f1 system, which is small enough to be easily loaded onto various vectors for cellular delivery, has gained attention. In this study, we explored the influence of temperature conditions on multiplex genome editing using CRISPR-Cas12f1 in an Escherichia coli model. It was revealed that when two distinct targets in the genome are edited simultaneously, the editing efficiency can be enhanced by allowing cells to recover at a reduced temperature during the editing process. Additionally, employing 3'-end truncated sgRNAs facilitated the simultaneous single-nucleotide level editing of three targets. Our results underscore the potential of optimizing recovery temperature and sgRNA design protocols in developing more effective and precise strategies for multiplex genome editing across various organisms.

Keywords

Acknowledgement

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00342735), and the Chung-Ang University research grant in 2023.

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