Improved HTGTS for CRISPR/Cas9 off-target detection

Jianhang Yin; Mengzhu Liu; Yang Liu; Jiazhi Hu

doi:10.21769/BioProtoc.3229

Improved HTGTS for CRISPR/Cas9 off-target detection

Bio Protoc. 2019 May 5;9(9):e3229. doi: 10.21769/BioProtoc.3229.

Authors

Jianhang Yin^{1

2}, Mengzhu Liu¹, Yang Liu¹, Jiazhi Hu^{1

2}

Affiliations

¹ The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking University, Beijing, 100871, China.
² Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China.

Abstract

Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a "bait" DNA double-stranded break (DSB) to capture genome-wide "prey" DNA DSBs. Here, we described an improved HTGTS (iHTGTS) method, in which size-selection beads were used to enhance reaction efficiency and a new primer system was designed to be compatible with Illumina Hiseq sequencing. Compared with LAM-HTGTS, iHTGTS is lower cost and has much higher sensitivity for off-target detection in HEK293T, K562, U2OS and HCT116 cell lines. So we believe that iHTGTS is a powerful method for comprehensively assessing Cas9 off-target effect.

Keywords: CRISPR-Cas9; Chromosomal translocation; IHTGTS; LAM-HTGTS; Off-target activity.