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

Junhao FuQing LiXiaoyu LiuTianxiang TuXiujuan LvXidi YinJineng LvZongming SongJia QuJinwei ZhangJinsong LiFeng Gu
Published in: Nature communications (2021)
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.
Keyphrases
  • crispr cas
  • genome editing
  • endothelial cells
  • high throughput
  • genome wide
  • stem cells
  • copy number
  • single cell
  • dna methylation