Integrative Analysis of CRISPRCas9 Target Sites in the Human HBB Gene

Abstract EN

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.