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Development of Base Editors for Simultaneously Editing Multiple Loci in Lactococcus lactis.

Kairen Tian1,2,3, Xia Hong1, Manman Guo1

  • 1Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China.

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|September 6, 2022
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Summary

Researchers developed a new CRISPR-deaminase-assisted base editor (CRISPR-DBE) for efficient, simultaneous multi-gene editing in Lactococcus lactis. This robust system enables precise cytidine-to-thymidine and adenosine-to-guanosine conversions, advancing genetic manipulation in this key dairy species.

Keywords:
Cas9 varinatsLactococcus lactisadenosine deaminasebase editorcytidine deaminasegene editing

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Lactococcus lactis is a crucial dairy species and model organism, but simultaneous multi-locus genetic editing remains challenging.
  • Existing CRISPR-Cas9 systems offer robust genetic manipulation but face limitations in multiplex editing efficiency for endogenous loci.

Purpose of the Study:

  • To develop a novel, robust, and efficient system for simultaneous multi-locus editing in Lactococcus lactis.
  • To introduce a double-strand break-free CRISPR-deaminase-assisted base editor (CRISPR-DBE) for precise base conversions.

Main Methods:

  • Development of CRISPR-cytidine deaminase-assisted base editor (CRISPR-cDBE) for C-to-T and CRISPR-adenosine deaminase-assisted base editor (CRISPR-aDBE) for A-to-G conversions.
  • Application of CRISPR-DBE system using sgRNA targeting, including validation in L. lactis strain NZ9000 and an industrial strain F44.
  • Genome-wide bioinformatics analysis to assess gene inactivation scope and evaluate Cas9 variant preferences for protospacer adjacent motifs.

Main Results:

  • CRISPR-cDBE efficiently introduced C-to-T mutations, and CRISPR-aDBE achieved high-efficiency A-to-G conversions within a 5-nucleotide window.
  • Simultaneous inactivation of multiple genes was successfully achieved in L. lactis NZ9000 using a single plasmid.
  • The temperature-sensitive CRISPR-DBE plasmid was rapidly cured, enabling continuous gene editing, and effective editing was demonstrated in industrial L. lactis strain F44.

Conclusions:

  • The developed CRISPR-DBE system provides a powerful and efficient tool for simultaneous multi-locus editing in Lactococcus lactis.
  • This technology facilitates precise genetic modifications, including gene inactivation and base conversions, in both model and industrial strains.
  • The advancement holds significant potential for diverse industrial applications in dairy microbiology and biotechnology.