Engineering of cytosine base editors with DNA damage minimization and editing scope diversification

Bo Yuan; Shuqian Zhang; Liting Song; Jinlong Chen; Jixin Cao; Jiayi Qiu; Zilong Qiu; Jingqi Chen; Xing-Ming Zhao; Tian-Lin Cheng

doi:10.1093/nar/gkad855

Engineering of cytosine base editors with DNA damage minimization and editing scope diversification

Nucleic Acids Res. 2023 Nov 10;51(20):e105. doi: 10.1093/nar/gkad855.

Authors

Bo Yuan^{1

2}, Shuqian Zhang^{1

3}, Liting Song^{4

5}, Jinlong Chen¹, Jixin Cao⁴, Jiayi Qiu¹, Zilong Qiu^{2

5

6}, Jingqi Chen^{4

7

8}, Xing-Ming Zhao^{4

7

8}, Tian-Lin Cheng¹

Affiliations

¹ Institute of Pediatrics, National Children's Medical Center, Children's Hospital, Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Fudan University, Shanghai 200032, China.
² Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China.
³ Department of Pediatrics, Qilu Hospital of Shandong University, Ji'nan 250012, China.
⁴ Institute of Science and Technology for Brain-inspired Intelligence, Key Laboratory of Computational Neuroscience and Brain-Inspired Intelligence, Fudan University, Shanghai 200433, China.
⁵ National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China.
⁶ Songjiang Hospital, Songjiang Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
⁷ State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Fudan University, Shanghai, China.
⁸ MOE Key Laboratory of Computational Neuroscience and Brain-Inspired Intelligence, and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China.

Abstract

Cytosine base editors (CBEs), which enable precise C-to-T substitutions, have been restricted by potential safety risks, including DNA off-target edits, RNA off-target edits and additional genotoxicity such as DNA damages induced by double-strand breaks (DSBs). Though DNA and RNA off-target edits have been ameliorated via various strategies, evaluation and minimization of DSB-associated DNA damage risks for most CBEs remain to be resolved. Here we demonstrate that YE1, an engineered CBE variant with minimized DNA and RNA off-target edits, could induce prominent DSB-associated DNA damage risks, manifested as γH2AX accumulation in human cells. We then perform deaminase engineering for two deaminases lamprey LjCDA1 and human APOBEC3A, and generate divergent CBE variants with eliminated DSB-associated DNA damage risks, in addition to minimized DNA/RNA off-target edits. Furthermore, the editing scopes and sequence preferences of APOBEC3A-derived CBEs could be further diversified by internal fusion strategy. Taken together, this study provides updated evaluation platform for DSB-associated DNA damage risks of CBEs and further generates a series of safer toolkits with diversified editing signatures to expand their applications.

Publication types

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

MeSH terms

CRISPR-Cas Systems
Cytosine*
DNA / genetics
DNA Damage
Gene Editing*
Humans
RNA / genetics

Substances

APOBEC3A protein, human
Cytosine
RNA
DNA