Engineering of near-PAMless adenine base editor with enhanced editing activity and reduced off-target

Xiaofang Cao; Junfan Guo; Shisheng Huang; Wenxia Yu; Guanglei Li; Lisha An; Xiangyang Li; Wanyu Tao; Qing Liu; Xingxu Huang; Xiaohua Jin; Xu Ma

doi:10.1016/j.omtn.2022.04.032

Engineering of near-PAMless adenine base editor with enhanced editing activity and reduced off-target

Mol Ther Nucleic Acids. 2022 May 4:28:732-742. doi: 10.1016/j.omtn.2022.04.032. eCollection 2022 Jun 14.

Authors

Xiaofang Cao^{1

2

3}, Junfan Guo^{4

5}, Shisheng Huang^{4

5}, Wenxia Yu^{4

5}, Guanglei Li⁴, Lisha An^{1

3}, Xiangyang Li^{4

5}, Wanyu Tao^{4

5}, Qing Liu⁶, Xingxu Huang⁴, Xiaohua Jin^{1

2

3}, Xu Ma^{1

2

3}

Affiliations

¹ National Research Institute for Family Planning, Beijing 100081, China.
² Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100005, China.
³ National Human Genetic Resources Center, Beijing 102206, China.
⁴ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
⁵ University of Chinese Academy of Sciences, Beijing 100049, China.
⁶ Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China.

Abstract

About 47% of pathogenic point mutations could be corrected by ABE-induced A·T-to-G·C conversions. However, the applications of ABEs are still hindered by undesired editing efficiency, limited editing scopes, and off-targeting effects. Here, we develop a new adenine base editor, by embedding TadA-8e monomer into SpRY-nCas9, named as CE-8e-SpRY, which exhibits higher activity at NRN than NYN PAMs favored by SpRY nuclease. CE-8e-SpRY could target nearly all genomic sites in principle and induces the highest targeting efficiency among tested SpRY-based ABEs. In addition, CE-8e-SpRY also shows reduced RNA and DNA off-targeting activities. With optimized sgRNAs, CE-8e-SpRY induces efficient or desired target editing at some disease-relevant loci where conventional ABEs were unable to induce precise and satisfied editing. Taken together, our CE-8e-SpRY could broaden the applicability of ABEs in correcting or introducing pathogenic point mutations.

Keywords: MT: RNA/DNA editing; SpRY-Cas9; adenine base editor; off-target; on-target; targeting scope.