Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310019, China.
Department of Biochemistry and Molecular Biology, Zhejiang University School of Medicine, Hangzhou 310058, China.
J Zhejiang Univ Sci B. 2021 Jan 15;22(1):73-86. doi: 10.1631/jzus.B2000282.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms, and holds tremendous promise in clinical applications. The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA (sgRNA) at a given site. However, little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks (DSBs). We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target, contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing. Here, we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing, and the opportunity this presents to optimize Cas9-based technology.
成簇规律间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 广泛用于细胞和生物体内的靶向基因组和表观基因组修饰和成像，在临床应用中具有巨大的潜力。该技术的效率和准确性部分取决于 Cas9-单指导 RNA（sgRNA）在给定位置的靶结合亲和力和停留时间。然而，人们很少关注靶结合亲和力和停留时间对 Cas9 诱导的 DNA 双链断裂（DSB）修复的影响。我们提出，Cas9-sgRNA 在切割靶标上的结合亲和力和停留时间的变化可能会改变 DSB 修复途径的选择，导致 CRISPR/Cas9 基因组编辑中出现显著异质性的突变。在这里，我们讨论了 Cas9-sgRNA 靶结合和停留对 CRISPR/Cas9 基因组编辑中 DSB 修复途径选择的影响，以及这为优化基于 Cas9 的技术提供了机会。
