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Construction and optimization of a base editor based on the MS2 system.

Shuzhen Zhang1, Songjie Feng2, Wen Jiang1

  • 1Department of Hematology, Southwest Hospital Third Military Medical University (Army Medical University) Chongqing China.

Animal Models and Experimental Medicine
|November 28, 2019
PubMed
Summary

This study developed an MS2-enhanced base editor for simultaneous multiple base editing at target genomic sites. The optimal MS2-BE-rAPOBEC1 system efficiently achieved simultaneous C-T mutations, offering a novel tool for genome function studies.

Keywords:
MS2‐MCPbase editingfunctional screeningsimultaneously multiple mutagenesis

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Area of Science:

  • Molecular Biology
  • Genome Engineering
  • Biotechnology

Background:

  • Catalytic-defective Cas9-cytosine deaminase fusions are key tools for base editing.
  • MS2 binding sites (MBS) recruit MS2 coat proteins (MCP) for signal amplification.
  • This study integrates MS2 amplification with base editing for simultaneous multi-base genomic modification.

Purpose of the Study:

  • To develop and optimize an MS2 signal amplification system for base editing.
  • To achieve simultaneous mutations at multiple target genomic sites.
  • To enhance the efficiency and window of base editing tools.

Main Methods:

  • Ligated MS2 binding sites to sgRNA 3'-end; fused MCP to cytosine deaminase 5'-end.
  • Recruited cytosine deaminase via MS2-MCP interaction for C-T base substitutions.
  • Tested various Cas9 variants, cytosine deaminases, and MS2 copy numbers.

Main Results:

  • dCas9 showed higher base mutation efficiency compared to nCas9 variants.
  • rAPOBEC1 deaminase was more efficient than AID.
  • Increasing MS2 copy numbers from 2x to 12x did not improve editing efficiency or window.

Conclusions:

  • An optimal base editor, MS2-BE-rAPOBEC1 (using sgRNA-2x MS2, MCP-rAPOBEC1, and dCas9), was developed.
  • This tool enables simultaneous mutation of multiple bases at target sites.
  • Provides a novel approach for investigating genome functions.