Reduced off-target effect of NG-BE4max by using NG-HiFi system

Huanhuan Shan; Zhiquan Liu; Yingqi Jia; Siyu Chen; Mao Chen; Yuning Song; Tingting Sui; Liangxue Lai; Zhanjun Li

doi:10.1016/j.omtn.2021.05.012

Reduced off-target effect of NG-BE4max by using NG-HiFi system

Mol Ther Nucleic Acids. 2021 May 19:25:168-172. doi: 10.1016/j.omtn.2021.05.012. eCollection 2021 Sep 3.

Authors

Huanhuan Shan¹, Zhiquan Liu¹, Yingqi Jia¹, Siyu Chen¹, Mao Chen¹, Yuning Song¹, Tingting Sui¹, Liangxue Lai^{1

2

3

4}, Zhanjun Li¹

Affiliations

¹ Key Laboratory of Zoonosis Research, Ministry of Education, Jilin University, Changchun 130062, China.
² CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
³ Guangzhou Regenerative Medicine and Health Guang Dong Laboratory (GRMH-GDL), Guangzhou 510005, China.
⁴ Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China.

Abstract

Recently, a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize a minimal NG protospacer adjacent motif (PAM) was reported to expand the targeting scope in genome editing. However, increased genome-wide off-target mutations with this variant compared with SpCas9 were reported in previous studies. In addition, lower base editing frequencies and higher unintended off-target mutations were also found in Hoxc13-ablated rabbits generated by NG-BE4max in our study. Here, a high-fidelity base editor, NG-HiFi, in comparison to NG-BE4max, showed retention of on-target activity while exhibiting significantly decreased off-target activity in Hoxc13-ablated rabbits. Collectively, the improved specificity and reduced off-target effect of SpCas9-NG assisted in cytidine base editing with the NG-HiFi system, providing a promising tool to precisely model human diseases in rabbits.

Keywords: Hoxc13; NG-BE4max; NG-HiFi; SpCas9-NG; off-target; rabbit.