德国分子生物学研究所Vassilis Roukos小组的最新研究，利用BreakTag将CRISPR-Cas9双链断裂图谱与基因编辑精度关联起来。2024年5月13日出版的《自然—生物技术》发表了这项成果。

据了解，Cas9能以钝化和交错两种方式裂解DNA，从而产生不同的编辑效果，但在很大程度上Cas9切口类型的决定因素是未知的。

在本研究中，研究人员开发了一种多功能方法-BreakTag，用于分析Cas9诱导的DNA双链断裂（DSB）并确定Cas9切口的决定因素。总体而言，在约3500个单导 RNA 靶向的15万多个内源性靶点和靶外位点，研究检测了SpCas9介导的裂解情况。研究发现， SpCas9 介导的DSB约35%是交错的，切口类型受DNA:gRNA互补性和工程Cas9变体的影响。

机器学习模型显示，Cas9的切口取决于原间隔序列，人类基因变异会影响Cas9切口的类型和DSB修复结果。Cas9和工程变体切口与修复结果的匹配数据集表明，Cas9介导的交错断裂与精确、模板化和可预测的单核苷酸插入有关，这表明基于裂解的gRNA设计可用于纠正临床相关致病性单核苷酸缺失。

附：英文原文

Title: Linking CRISPR–Cas9 double-strand break profiles to gene editing precision with BreakTag

Author: Longo, Gabriel M. C., Sayols, Sergi, Kotini, Andriana G., Heinen, Sabine, Mckel, Martin M., Beli, Petra, Roukos, Vassilis

Issue&Volume: 2024-05-13

Abstract: Cas9 can cleave DNA in both blunt and staggered configurations, resulting in distinct editing outcomes, but what dictates the type of Cas9 incisions is largely unknown. In this study, we developed BreakTag, a versatile method for profiling Cas9-induced DNA double-strand breaks (DSBs) and identifying the determinants of Cas9 incisions. Overall, we assessed cleavage by SpCas9 at more than 150,000 endogenous on-target and off-target sites targeted by approximately 3,500 single guide RNAs. We found that approximately 35% of SpCas9 DSBs are staggered, and the type of incision is influenced by DNA:gRNA complementarity and the use of engineered Cas9 variants. A machine learning model shows that Cas9 incision is dependent on the protospacer sequence and that human genetic variation impacts the configuration of Cas9 cuts and the DSB repair outcome. Matched datasets of Cas9 and engineered variant incisions with repair outcomes show that Cas9-mediated staggered breaks are linked with precise, templated and predictable single-nucleotide insertions, demonstrating that a scission-based gRNA design can be used to correct clinically relevant pathogenic single-nucleotide deletions.

DOI: 10.1038/s41587-024-02238-8

Source: https://www.nature.com/articles/s41587-024-02238-8