Computational Analysis Concerning the Impact of DNA Accessibility on CRISPR-Cas9 Cleavage Efficiency
Computational Analysis Concerning the Impact of DNA Accessibility on CRISPR-Cas9 Cleavage Efficiency
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DOI：
10.1016/j.ymthe.2019.10.008
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发表时间：
2020-01-08
影响因子：
12.4
通讯作者：
Dampier, Will
中科院分区：
文献类型：
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作者：
Chung, Cheng-Han;Allen, Alexander G.;Dampier, Will
定义影响CRISPR/ Cas9特异性的变量一直是主要的研究焦点。尽管指导RNA和靶DNA之间的序列互补性基本上决定了切割效率，但靶向基因座的DNA可及性也被假设为重要因素。在这项研究中，功能数据来自两个全基因组检测，全基因组，无偏鉴定DSB测序使之成为可能，（GUIDE-seq）和环化用于通过测序体外报告切割作用（CIRCLE-seq），已经结合DNA可及性进行了计算分析，所述DNA可及性通过DNA酶I-超敏测序从DNA元件百科全书（ENCODE）确定数据库和来自Sequence Read Archive的转录组，以确定细胞因子是否影响CRISPR诱导的切割效率。选择CIRCLE-seq和GUIDE-seq数据集分别代表细胞因子的不存在和存在。数据分析显示，序列相似性和CRISPR诱导的切割频率之间的相关性被调节DNA可及性水平的细胞因子的存在所改变。当切割位点位于不易接近的区域时，上述相关性被消除。此外，CRISPR介导的编辑即使在不足以表达大多数内源基因的区域也是允许的。这些结果为剖析局部染色质调节标记物对CRISPR诱导的切割效率的贡献提供了强有力的基础。
Defining the variables that impact the specificity of CRISPR/ Cas9 has been a major research focus. Whereas sequence complementarity between guide RNA and target DNA substantially dictates cleavage efficiency, DNA accessibility of the targeted loci has also been hypothesized to be an important factor. In this study, functional data from two genome-wide assays, genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq) and circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), have been computationally analyzed in conjunction with DNA accessibility determined via DNase I-hypersensitive sequencing from the Encyclopedia of DNA Elements (ENCODE) Database and transcriptome from the Sequence Read Archive to determine whether cellular factors influence CRISPR-induced cleavage efficiency. CIRCLE-seq and GUIDE-seq datasets were selected to represent the absence and presence of cellular factors, respectively. Data analysis revealed that correlations between sequence similarity and CRISPR-induced cleavage frequency were altered by the presence of cellular factors that modulated the level of DNA accessibility. The above-mentioned correlation was abolished when cleavage sites were located in less accessible regions. Furthermore, CRISPR-mediated edits were permissive even at regions that were insufficient for most endogenous genes to be expressed. These results provide a strong basis to dissect the contribution of local chromatin modulation markers on CRISPR-induced cleavage efficiency.