Marin-Gonzalez Alberto, Rybczynski Adam T, Zou Roger S, Ha Taekjip
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, United States; Department of Pediatrics, Harvard Medical School, Boston, MA, United States; Howard Hughes Medical Institute, Boston, MA, United States.
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, United States; Department of Pediatrics, Harvard Medical School, Boston, MA, United States.
Methods Enzymol. 2025;712:1-22. doi: 10.1016/bs.mie.2025.01.011. Epub 2025 Feb 7.
A mechanistic understanding of the different pathways involved in the repair of DSBs is a timely, yet challenging task. CRISPR-Cas9 is a powerful tool to induce DNA double-strand breaks (DSB) at defined genomic locations to study the ensuing repair response, but Cas9 studies are typically limited by i) low-throughput induction of DSB, by targeting only one or a few genomic sites, or ii) the use of genetically integrated reporter systems, which do not always reflect endogenous phenotypes. To address these limitations, we developed multi-target CRISPR, a Cas9-based tool to controllably induce DSBs in high-throughput at endogenous sites, by leveraging repetitive genomic regions. In this Chapter, we describe how to design and execute a multi-target CRISPR experiment. We also detail how to analyze next-generation sequencing data for characterization of DSB repair events at multiple cut sites. We envision that multi-target CRISPR will become a valuable tool for the study of mammalian DSB repair mechanisms.
深入理解DNA双链断裂（DSB）修复过程中涉及的不同途径是一项既紧迫又具有挑战性的任务。CRISPR-Cas9是一种强大的工具，可在特定基因组位置诱导DNA双链断裂（DSB），以研究随后的修复反应，但Cas9研究通常受到以下限制：i）通过仅靶向一个或几个基因组位点进行低通量DSB诱导；ii）使用基因整合报告系统，该系统并不总是能反映内源性表型。为了解决这些限制，我们开发了多靶点CRISPR，这是一种基于Cas9的工具，通过利用重复基因组区域，可在内源性位点以高通量方式可控地诱导DSB。在本章中，我们将描述如何设计和执行多靶点CRISPR实验。我们还将详细介绍如何分析下一代测序数据，以表征多个切割位点的DSB修复事件。我们预计多靶点CRISPR将成为研究哺乳动物DSB修复机制的宝贵工具。
