Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease
Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease
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DOI：
10.1038/nbt.2916
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发表时间：
2014-07-01
影响因子：
46.9
通讯作者：
Adli, Mazhar
中科院分区：
文献类型：
--
作者：
Kuscu, Cem;Arslan, Sevki;Adli, Mazhar
使用CRISPR-Cas9系统进行RNA引导的基因组编辑在基础和临床研究中具有巨大的潜力，但靶向特异性的决定因素和脱靶切割的程度仍然没有得到充分的理解。使用染色质免疫沉淀和高通量测序（ChIP-seq），我们在HEK 293 T细胞中绘制了无催化活性Cas9（dCas 9）的全基因组结合位点，并结合了12种不同的单向导RNA（sgRNA）。取决于sgRNA，dCas 9结合的脱靶位点的数目从近似10个变化到> 1，000个。脱靶结合位点的分析显示了sgRNA引导序列的PAM近端区域的重要性，并且dCas 9结合位点在开放的染色质区域中富集。当用催化活性Cas9靶向时，一些脱靶结合位点在ChIP-seq峰周围的区域中具有高于背景水平的插入缺失，但通常以低于中靶位点的速率。我们的结果阐明了Cas9靶向的主要决定因素，并且我们表明ChIP-seq允许在全基因组范围内无偏地检测Cas9结合位点。
RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from similar to 10 to > 1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide.