TadA reprogramming to generate potent miniature base editors with high precision

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

doi:10.1038/s41467-023-36004-2

TadA reprogramming to generate potent miniature base editors with high precision

Nat Commun. 2023 Jan 26;14(1):413. doi: 10.1038/s41467-023-36004-2.

Authors

Shuqian Zhang^#^{1

2}, Liting Song^#³, Bo Yuan^#⁴, Cheng Zhang^#¹, Jixin Cao^#³, Jinlong Chen¹, Jiayi Qiu¹, Yilin Tai⁵, Jingqi Chen^{3

6

7}, Zilong Qiu^{8

9

10}, Xing-Ming Zhao^{11

12

13}, Tian-Lin Cheng¹⁴

Affiliations

¹ Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Institute of Pediatrics, National Children's Medical Center, Children's Hospital, Fudan University, Shanghai, China.
² Department of Pediatrics, Qilu Hospital of Shandong University, Ji'nan, 250012, China.
³ Institute of Science and Technology for Brain-Inspired Intelligence, Fudan University, Shanghai, China.
⁴ 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.
⁵ Institutes of Brain Science, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Fudan University, Shanghai, 200032, China.
⁶ State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Fudan University, Shanghai, China.
⁷ MOE Key Laboratory of Computational Neuroscience and Brain-Inspired Intelligence, and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China.
⁸ 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. zqiu@ion.ac.cn.
⁹ National Clinical Research Center for Aging and Medicine, Huashan Hopsital, Fudan University, Shanghai, 200032, China. zqiu@ion.ac.cn.
¹⁰ Songjiang Hospital, Songjiang Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, China. zqiu@ion.ac.cn.
¹¹ Institute of Science and Technology for Brain-Inspired Intelligence, Fudan University, Shanghai, China. xmzhao@fudan.edu.cn.
¹² State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Fudan University, Shanghai, China. xmzhao@fudan.edu.cn.
¹³ MOE Key Laboratory of Computational Neuroscience and Brain-Inspired Intelligence, and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China. xmzhao@fudan.edu.cn.
¹⁴ Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Institute of Pediatrics, National Children's Medical Center, Children's Hospital, Fudan University, Shanghai, China. chengtianlin@fudan.edu.cn.

^# Contributed equally.

Abstract

Although miniature CRISPR-Cas12f systems were recently developed, the editing efficacy and targeting range of derived miniature cytosine and adenine base editors (miniCBEs and miniABEs) have not been comprehensively addressed. Moreover, functional miniCBEs have not yet be established. Here we generate various Cas12f-derived miniCBEs and miniABEs with improved editing activities and diversified targeting scopes. We reveal that miniCBEs generated with traditional cytidine deaminases exhibit wide editing windows and high off-targeting effects. To improve the editing signatures of classical CBEs and derived miniCBEs, we engineer TadA deaminase with mutagenesis screening to generate potent miniCBEs with high precision and minimized off-target effects. We show that newly designed miniCBEs and miniABEs are able to correct pathogenic mutations in cell lines and introduce genetic mutations efficiently via adeno-associated virus delivery in the brain in vivo. Together, this study provides alternative strategies for CBE development, expands the toolkits of miniCBEs and miniABEs and offers promising therapeutic tools for clinical applications.

Publication types

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

MeSH terms

CRISPR-Cas Systems* / genetics
Cytidine Deaminase / genetics
Cytidine Deaminase / metabolism
Cytosine / metabolism
Gene Editing*
Mutation

Substances

Cytidine Deaminase
Cytosine