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用多靶点CRISPR的大规模平行基因组扰动监测内源性位点的Cas9活性和DNA修复
作者:小柯机器人 发布时间:2022/9/8 16:51:41

美国约翰霍普金斯大学医学院Taekjip Ha团队近期取得重要工作进展,他们研究利用多靶点CRISPR的大规模平行基因组扰动监测内源性位点的Cas9活性和DNA修复。相关工作2022年9月5日在线发表于《自然—细胞生物学》杂志上。

研究人员提出了一种方法,该方法结合了CRISPR系统和高通量测序,CRISPR系统同时靶向数百个表观遗传多样化的内源基因组位点,用来大规模测量Cas9动态变化和细胞应答反应。这种大规模的CRISPR多路复用是通过多靶标导向RNA(mgRNAs)实现的,这种简并导向RNA将Cas9引导至预定数量的靶位点。mgRNA揭示了对Cas9结合和切割的普遍见解,揭示了切割后Cas9离开和修复因子在原间隔区相邻基序近端基因组DNA上的快速募集。

此外,通过绕过来自引导RNA序列的混杂效应,mgRNA揭示了在染色质可接近区域增强了Cas9结合,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进一步实现了细胞对双链断裂反应的高通量研究,具有高时间分辨率,揭示了双链断裂可逆的DNA损伤诱导的染色质解压缩的存在、程度(小于2kb)和动力学(~1 h)。总而言之,这项工作将mgRNAs建立为用于多路复用CRISPR的通用平台,并促进了研究人员对细胞内Cas9活性和内源性基因位点的DNA损伤反应的理解。

附:英文原文

Title: Massively parallel genomic perturbations with multi-target CRISPR interrogates Cas9 activity and DNA repair at endogenous sites

Author: Zou, Roger S., Marin-Gonzalez, Alberto, Liu, Yang, Liu, Hans B., Shen, Leo, Dveirin, Rachel K., Luo, Jay X. J., Kalhor, Reza, Ha, Taekjip

Issue&Volume: 2022-09-05

Abstract: 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 2kb) and kinetics (~1h) 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.

DOI: 10.1038/s41556-022-00975-z

Source: https://www.nature.com/articles/s41556-022-00975-z

期刊信息

Nature Cell Biology:《自然—细胞生物学》,创刊于1999年。隶属于施普林格·自然出版集团,最新IF:20.042
官方网址:https://www.nature.com/ncb/
投稿链接:https://mts-ncb.nature.com/cgi-bin/main.plex