摘要
Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the “double tap” method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a “second chance” at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.
摘要译文
可以通过操纵同源指导修复（HDR）途径来利用可编程的双链DNA断裂（DSB）进行精确基因组编辑。但是，最终连接修复途径通常胜过HDR，并在DSB站点引入碱基（Indels）的插入和缺失，从而降低了精度结果。已经表明，给定DSB位点的indel序列是可重现的，甚至可以预测。在这里，我们报告了一种一般策略（“双击”方法），以提高HDR介导的精确基因组编辑效率，以利用Indel序列的可重复性性质。该方法仅涉及多个GRNA的使用：靶向野生型基因组序列的主要GRNA，以及针对最常见的indel序列的一个或多个次级GRNA，实际上在HDR上提供了“第二次机会” - 介导的编辑。这项原则研究列出了双击方法，作为增强哺乳动物细胞精确编辑的简单但有效的选择。
Bodai; Zsolt[1];Bishop; Alena L.[2];Gantz; Valentino M.[2];Komor; Alexis C.[1]. Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies[J]. Nature Communications, 2022: 1-15