Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene.
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.
Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China.
Yumei
Y
Luo
Department of Biological Sciences, National University of Singapore, Singapore 117543.
Detu
0000-0002-9268-1012
D
Zhu
Department of Computer Science, National University of Singapore, Singapore 117543.
Zhizhuo
Z
Zhang
Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China.
Yaoyong
Y
Chen
Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China.
Xiaofang
X
Sun
D001665:Binding Sites; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D005091:Exons / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D055544:beta-Globins / Q000235:genetics* / Q000378:metabolism
D055544:beta-Globins