Massively parallel genomic perturbations with multi-target CRISPR interrogates Cas9 activity and DNA repair at endogenous sites.
Massively parallel genomic perturbations with multi-target CRISPR interrogates Cas9 activity and DNA repair at endogenous sites.
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使用多靶点CRISPR的大规模平行基因组扰动询问内源性位点的Cas9活性和DNA修复。
DOI：
10.1038/s41556-022-00975-z
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发表时间：
2022-09
影响因子：
21.3
通讯作者：
中科院分区：
文献类型：
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作者：
在这里，我们提出了一种方法，该方法结合了成簇的规则间隔短回文重复序列（CRISPR）系统，该系统同时靶向数百个表观遗传多样性内源基因组位点，并具有高通量测序，以大规模测量Cas9动力学和细胞反应。CRISPR的这种大规模多重化是通过多靶向导RNA（mgRNA）实现的，所述多靶向导RNA是将Cas9引导到预定数量的良好定位的位点的简并向导RNA。mgRNA揭示了对Cas9结合和切割的可概括的见解，揭示了在原型间隔区邻近基序近端基因组DNA处的快速切割后Cas9离开和修复因子加载。此外，通过绕过来自指导RNA序列的混淆效应，mgRNA揭示了Cas9结合在染色质可接近区域增强，并且结合的Cas9的切割在转录区域附近更有效。结合光介导的Cas9活性的激活和失活，mgRNA进一步实现了对具有高时间分辨率的双链断裂的细胞响应的高通量研究，揭示了可逆DNA损伤诱导的染色质解压缩的存在、程度（低于2kb）和动力学（约1小时）。总而言之，这项工作建立了mgRNA作为多重CRISPR的通用平台，并推进了我们对细胞内Cas9活性和内源性基因座DNA损伤反应的理解。Zou等人使用单个多靶向导RNA将Cas9引导至通过高通量短读段测序映射的大量基因座。这种多靶点CRISPR系统允许对基因组编辑和DNA修复进行详细研究。
Here we present an approach that combines a clustered regularly interspaced short palindromic repeats (CRISPR) system that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of multi-target guide RNAs (mgRNAs), degenerate guide RNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, revealing rapid post-cleavage Cas9 departure and repair factor loading at protospacer adjacent motif-proximal genomic DNA. Moreover, by bypassing confounding effects from guide RNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and cleavage by bound Cas9 is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double-strand breaks with high temporal resolution, revealing the presence, extent (under 2 kb) and kinetics (~1 h) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci. Zou et al. use a single multi-target guide RNA to direct Cas9 to a high number of loci mapped by high-throughput short-read sequencing. This multi-target CRISPR system allows detailed studies of genome editing and DNA repair.
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