GUIDE-Seq for TRAC sgRNAs

GUIDE-Seq was performed of two CRISPR-Cas9 single guide RNAs used to target an Streptococcus pyogenes Cas9 nuclease (SpCas9) to the TRAC gene locus. The two sgRNAs differ in one position of their respective target-specific spacer sequence (TRAC Wiebking 2020: 5-GAGAATCAAAATCGGTGAAT-3; TRAC Charite: 5- GGGAATCAAAATCGGTGAAT-3). The study was performed to compare the location and the amount of putative off-targets sites of the respective sgRNAs in SpCas9-overexpressing HEK293 cells.

本研究针对两款用于将化脓性链球菌Cas9（Streptococcus pyogenes Cas9, SpCas9）靶向至TRAC（T细胞受体α链恒定区）基因座的CRISPR-Cas9单引导RNA（single guide RNA, sgRNA）开展了GUIDE-Seq实验。两款sgRNA的靶特异性间隔序列仅存在一处碱基差异：TRAC Wiebking 2020对应的序列为5'-GAGAATCAAAATCGGTGAAT-3'，TRAC Charite对应的序列为5'-GGGAATCAAAATCGGTGAAT-3'。本研究旨在对比过表达SpCas9的HEK293细胞中，两款sgRNA的潜在脱靶位点的位置与数量。