Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; University of Chinese Academy of Sciences, Beijing 100049, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China.
Mol Ther. 2023 Apr 5;31(4):1167-1176. doi: 10.1016/j.ymthe.2023.01.028. Epub 2023 Feb 1.
Imperfect -gRNA (igRNA) provides a simple strategy for single-base editing of a base editor. However, a significant number of igRNAs need to be generated and tested for each target locus to achieve efficient single-base reversion of pathogenic single nucleotide variations (SNVs), which hinders the direct application of this technology. To provide ready-to-use igRNAs for single-base and bystander-less correction of all the adenine base editor (ABE)-reversible pathogenic SNVs, we employed a high-throughput method to edit all 5,253 known ABE-reversible pathogenic SNVs, each with multiple systematically designed igRNAs, and two libraries of 96,000 igRNAs were tested. A total of 1,988 SNV loci could be single-base reversed by igRNA with a >30% efficiency. Among these 1,988 loci, 378 SNV loci exhibited an efficiency of more than 90%. At the same time, the bystander editing efficiency of 76.62% of the SNV loci was reduced to 0%, while remaining below 1% for another 18.93% of the loci. These ready-to-use igRNAs provided the best solutions for a substantial portion of the 4,657 pathogenic/likely pathogenic SNVs. In this work, we overcame one of the most significant obstacles of base editors and provide a ready-to-use platform for the genetic treatment of diseases caused by ABE-reversible SNVs.
不完美的引导 RNA (igRNA) 为碱基编辑器的单碱基编辑提供了一种简单的策略。然而，为了实现对致病单核苷酸变异 (SNV) 的高效单碱基回复，需要针对每个靶标位点生成和测试大量的 igRNA，这阻碍了该技术的直接应用。为了提供用于单碱基和无旁观者校正的即用型 igRNA，我们采用高通量方法编辑了所有 5253 个已知的碱基编辑器 (ABE) 可逆致病性 SNV，每个 SNV 都设计了多个有系统的 igRNA，并测试了两个 96000 个 igRNA 的文库。共有 1988 个 SNV 位点可以通过 igRNA 以 >30%的效率进行单碱基反转。在这 1988 个位点中，有 378 个 SNV 位点的效率超过 90%。同时，76.62%的 SNV 位点的旁观者编辑效率降低到 0%，而另外 18.93%的位点的旁观者编辑效率仍然低于 1%。这些即用型 igRNA 为 4657 个致病性/可能致病性 SNV 中的大部分提供了最佳解决方案。在这项工作中，我们克服了碱基编辑器的最大障碍之一，为 ABE 可逆 SNV 引起的疾病的遗传治疗提供了一个即用型平台。
