Improved HTGTS for CRISPR/Cas9 Off-target Detection
Jianhang Yin,
Mengzhu Liu,
Yang Liu,
Jiazhi Hu
Affiliations
Jianhang Yin
The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking University, Beijing, 100871, ChinaPeking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China
Mengzhu Liu
The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center,, School of Life Sciences, Peking University, Beijing, 100871, China
Yang Liu
The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking University, Beijing, 100871, China
Jiazhi Hu
The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center,, School of Life Sciences, Peking University, Beijing, 100871, ChinaPeking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China
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.
