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

Abstract

Abstract Background Catalytic defect Cas9‐cytosine deaminase fusion is widely used in base editing. The Multiple copy numbers of the MS2 binding site (MBS) can recruit multiple MS2 coat proteins (MCPs), which are usually applied to amplify signals. Our study aimed to apply the MS2 signal amplification system to the base editing system in order to achieve simultaneous mutations of multiple bases at the target genome site. Methods Multiple copy numbers of the MS2 were ligated to the 3′‐end of sgRNA, and MCP was fused to the 5′‐end of cytosine deaminases. The MS2 was recognized by MCP to recruit cytosine deaminase for base substitutions (C‐T) at the target site. Different Cas9 variants, different cytosine deaminases and different copy numbers of MS2 were tested in this system, and the different versions of base editors were compared by editing efficiency and window. Results In this study, dCas9, nCas9 (D10A) and nCas9 (H840A) were used. Among these 3 Cas9 variants, dCas9 exhibited higher base mutation efficiency. Two cytosine deaminases were then applied and the efficiency of rAPOBEC1 deaminase was found to be higher than AID. We also increased the copy numbers of MS2 linked to sgRNA from 2 to 12. Disappointingly, the sgRNA‐12x MS2 did not improve the editing efficiency or increase the editing window. Conclusion An optimal version of base editor based on the MS2 system, MS2‐BE‐rAPOBEC1 (sgRNA‐2x MS2, MCP‐rAPOBEC1 and dCas9), was obtained. This tool can simultaneously mutate multiple bases at the target site, providing a new approach for the study of genome functions.

Keywords

• base editing
• functional screening
• MS2‐MCP
• simultaneously multiple mutagenesis