聚集的定期间隔短回文重复(CRISPR)和CRISPR相关(Cas)系统是古细菌和细菌中用于识别和破坏外部入侵者DNA的免疫防御系统。来自化脓性链球菌(SpCas9)的CRISPR-SpCas9系统已成为最广泛使用的基因组编辑工具,并显示出临床应用的希望。然而,脱靶效应仍然是CRISPR-SpCas9基因组编辑的主要挑战.基于对结构和裂解程序的分析,我们提出了两种策略来修改SpCas9结构并减少脱靶效应。缩短HNH或REC3接头(策略#1)旨在将HNH或REC3的主要位置远离单向导RNA(sgRNA)/DNA杂交(杂交),同时延长sgRNA周围的螺旋(策略#2)旨在加强SpCas9和sgRNA/DNA之间的接触。我们设计了11个SpCas9变体(变体No.1-变体No.11),并验证了它们在经典基因组位点EMX1-1,EMX1-1-OT1和EMX1-1-OT2上的效率。前三个有效的SpCas9变体,变异1号,变异2号和变异5号在其他基因组位点进行了额外验证.将进一步选择的变体No.1与之前的两个SpCas9变体进行比较,HypaCas9(2017年发布的超精确Cas9变体)和eSpCas9(1.1)(2016年发布的“增强特异性”SpCas9变体)。在两个基因组位点上,EMX1-1和FANCF-1。结果表明,Thr769和Gly906的缺失可以显著降低脱靶效应,同时在大多数选定的基因组位点保持稳健的中靶效率。
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems are immunological defenses used in archaea and bacteria to recognize and destroy DNA from external invaders. The CRISPR-
SpCas9 system harnessed from Streptococcus pyogenes (
SpCas9) has become the most widely utilized genome editing tool and shows promise for clinical application. However, the off-target effect is still the major challenge for the genome editing of CRISPR-
SpCas9. Based on analysis of the structure and cleavage procedures, we proposed two strategies to modify the
SpCas9 structure and reduce off-target effects. Shortening the HNH or REC3 linkers (Strategy #1) aimed to move the primary position of HNH or REC3 far away from the single-guide RNA (sgRNA)/DNA hybrid (hybrid), while elongating the helix around the sgRNA (Strategy #2) aimed to strengthen the contacts between SpCas9 and the sgRNA/DNA. We designed 11 SpCas9 variants (variant No.1- variant No.11) and verified their efficiencies on the classic genome site EMX1-1, EMX1-1-OT1, and EMX1-1-OT2. The top three effective
SpCas9 variants, variant No.1, variant No.2, and variant No.5, were additionally validated on other genome sites. The further selected variant No.1 was compared with two previous SpCas9 variants, HypaCas9 (a hyper-accurate Cas9 variant released in 2017) and eSpCas9 (1.1) (an \"enhanced specificity\"
SpCas9 variant released in 2016), on two genome sites, EMX1-1 and FANCF-1. The results revealed that the deletion of Thr769 and Gly906 could substantially decrease off-target effects, while maintaining robust on-target efficiency in most of the selected genome sites.