Enhancing glycosylase base-editor activity by fusion to transactivation modules

Xingxiao Dong; Chao Yang; Zhenzhen Ma; Ming Chen; Xueli Zhang; Changhao Bi

doi:10.1016/j.celrep.2022.111090

Enhancing glycosylase base-editor activity by fusion to transactivation modules

Cell Rep. 2022 Jul 19;40(3):111090. doi: 10.1016/j.celrep.2022.111090.

Authors

Xingxiao Dong¹, Chao Yang², Zhenzhen Ma³, Ming Chen⁴, Xueli Zhang⁵, Changhao Bi⁶

Affiliations

¹ School of Biological Engineering, Dalian Polytechnic University, Dalian, China.
² Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
³ College of Life Sciences, Nankai University, Tianjin, China.
⁴ School of Biological Engineering, Dalian Polytechnic University, Dalian, China. Electronic address: chenming@dlpu.edu.cn.
⁵ Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China. Electronic address: zhang_xl@tib.cas.cn.
⁶ Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China. Electronic address: bi_ch@tib.cas.cn.

PMID: 35858572
DOI: 10.1016/j.celrep.2022.111090

Abstract

Base editors (BEs) are a group of genetic tools with potential in both scientific and medical research. Recently, a glycosylase BE (GBE), which converts C to G, has been constructed. However, the editing efficiency and targeting scope remains to be further exploited. Here, we renovate the GBE by first fusing it to various transactivation modules including Vp64, leading to a higher conversion of C to G relative to GBE in HEK293T cells. Further, higher editing efficiency, enhanced editing purity, and an enlarged editing window are acquired by the combination of SunTag system, GBE, and VP64. Finally, a SpRY-Cas9 variant is used to expand the targeting scope for Vp64-GBE. Vp64-SpRY-GBE and SpRY-GBE target genomic sites with non-NGG PAM, and Vp64-SpRY-GBE demonstrates better performance compared with SpRY-GBE. The construction of GBE variants with superior performance and versatile editing scope broadens the toolbox of BEs and may contribute to genetic therapies with C-to-G mutation.

Keywords: CP: Molecular biology; CRISPR-Cas9; base editor; glycosylase base editor; transactivation modules.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems* / genetics
Gene Editing*
HEK293 Cells
Humans
Mutation / genetics
Transcriptional Activation / genetics