Human cell based directed evolution of adenine base editors with improved efficiency

Junhao Fu, Qing Li, Xiaoyu Liu, Tianxiang Tu, Xiujuan Lv, Xidi Yin, Jineng Lv, Zongming Song, Jia Qu, Jinwei Zhang, Jinsong Li () and Feng Gu ()
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Junhao Fu: Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry
Qing Li: Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences
Xiaoyu Liu: Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry
Tianxiang Tu: Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry
Xiujuan Lv: Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry
Xidi Yin: Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences
Jineng Lv: Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry
Zongming Song: Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry
Jia Qu: Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry
Jinwei Zhang: National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
Jinsong Li: Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences
Feng Gu: Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry

Nature Communications, 2021, vol. 12, issue 1, 1-11

Abstract: Abstract Adenine base editors (ABE) are genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion. However, the low activity of ABE at certain sites remains a major bottleneck that precludes efficacious applications. Here, to address it, we develop a directional screening system in human cells to evolve the deaminase component of the ABE, and identify three high-activity NG-ABEmax variants: NG-ABEmax-SGK (R101S/D139G/E140K), NG-ABEmax-R (Q154R) and NG-ABEmax-K (N127K). With further engineering, we create a consolidated variant [NG-ABEmax-KR (N127K/Q154R)] which exhibit superior editing activity both in human cells and in mouse disease models, compared to the original NG-ABEmax. We also find that NG-ABEmax-KR efficiently introduce natural mutations in gamma globin gene promoters with more than four-fold increase in editing activity. This work provides a broadly applicable, rapidly deployable platform to directionally screen and evolve user-specified traits in base editors that extend beyond augmented editing activity.

Date: 2021
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DOI: 10.1038/s41467-021-26211-0

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