Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene
Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene
复制标题
人类 HBB 基因中 CRISPR/Cas9 靶位点的综合分析
DOI：
10.1155/2015/514709
复制
发表时间：
2015-01-01
影响因子：
--
通讯作者：
Sun, Xiaofang
中科院分区：
文献类型：
--
作者：
Luo, Yumei;Zhu, Detu;Sun, Xiaofang
最近，成簇的规则间隔短回文重复序列（CRISPR）系统已经成为一种强大的可定制人工核酸酶，以促进组织再生和同基因疾病建模的精确遗传校正。然而，先前的研究报道了CRISPR系统在人类细胞中的大量脱靶活性，并且大量推定的脱靶位点需要实验验证，因此激发了生物信息学方法来合理设计CRISPR系统并预测其潜在的脱靶效应。在这里，我们描述了一个综合分析过程，以确定人类β-珠蛋白基因（HBB）中的特定CRISPR靶位点，并预测其脱靶效应。我们的方法包括脱靶分析在编码和非编码区，这是被忽视的，以前的研究。发现内含子中的CRISPR靶位点在编码区中具有比外显子中更少的脱靶位点。值得注意的是，含有某些转录因子基序的靶位点在其脱靶集中富集了相关转录因子的结合位点。我们还发现内含子位点具有较少的SNP，这导致在临床应用期间不同个体中CRISPR效率的变化较小。我们的研究提供了一个标准的分析程序，以选择特定的CRISPR靶点进行遗传校正。
Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the human β-globin gene (HBB) and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction.