TY  - JOUR
TI  - Improved HTGTS for CRISPR/Cas9 Off-target Detection
AU  - Yin, Jianhang  
AU  - Liu, Mengzhu 
AU  - Liu, Yang  
AU  - Hu, Jiazhi 
VL  - 9
IS  - 9
PY  - 2019
DA  - 2019/05/05
SP  - e3229
C1  - Bio-protocol 2019;9:e3229
DO  - 10.21769/BioProtoc.3229
UR  - https://doi.org/10.21769/BioProtoc.3229
AB  - 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.
KW  - CRISPR-Cas9
KW  - Off-target activity
KW  - Chromosomal translocation
KW  - LAM-HTGTS
KW  - iHTGTS
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False