Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations

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Brief intro:

  • Author: Lijie Wang, Wei Xue, Hongxia Zhang, Runze Gao, Houyuan Qiu, Jia Wei, Lina Zhou, Yun-Ni Lei, Xiaocheng Wu, Xiao Li, Chengfang Liu, Jing Wu, Qiubing Chen, Hanhui Ma, Xingxu Huang, Cheguo Cai, Ying Zhang, Bei Yang, Hao Yin, Li Yang and Jia Chen
  • Journal: Nature Cell Biology
  • Doi: https://www.doi.org/10.1038/s41556-021-00671-4
  • Publication Date: 2021 May

Products/Services used in the paper

Quotation shows PackGene:The AAV8-nSpCas9 (D10A) and AAV8-tBE-V5-mA3 targeting PCSK9-E4 were provided by PackGene Biotech and the titres were 1 × 10^13 vg per ml.

Research Field:base editing system

AAV Serotype:AAV8

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Abstract

The fusion of CRISPR–Cas9 with cytidine deaminases leads to base editors (BEs) capable of programmable C-to-T editing, which has potential in clinical applications but suffers from off-target (OT) mutations. Here, we used a cleavable deoxycyti-dine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, the tBE remains inactive at OT sites with the fusion of a cleavable dCDI, therefore eliminating unintended mutations. When binding at on-target sites, the tBE is transformed to cleave off the dCDI domain and catalyses targeted deamination for precise base editing. After delivery into mice through a dual-adeno-associated virus (AAV) system, the tBE system created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9, resulting in a ~30–40% decrease in total cholesterol. The development of tBE establishes a highly specific base editing system and its in vivo efficacy has potential for therapeutic applications.

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