On/Off targets INDEL analysis by Next Generation Sequencing. On/Off targets INDEL analysis by Next Generation Sequencing

SpCas9 INDEL frequency was evaluated at both the on- and off-target sites of guideRNA targeting either the murine Rhodopsin or Albumin by Next Generation Sequencing. Minimal INDELs were found at the off target sites evaluted, while SpCas9 activity was predominantly on-target. Overall design: Three gRNA- and 2 scramble control-treated samples of either retina or liver were used to amplify the gRNA on-target and the top 10 off-target sites.

本研究通过下一代测序（Next Generation Sequencing），评估了靶向小鼠视紫红质（Rhodopsin）或白蛋白（Albumin）的向导RNA（guideRNA）的靶位点与脱靶位点处的SpCas9介导的插入缺失（INDEL）频率。在所检测的脱靶位点中仅发现极微量的插入缺失变异，且SpCas9的编辑活性主要集中于靶位点。实验整体设计：针对视网膜与肝脏组织，设置3组向导RNA处理样本及2组乱序对照（scramble control）样本，用于扩增向导RNA的靶位点及排名前十的脱靶位点。