Molecular Composite Physiology Research Group, Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Science City 305-8566, Japan.
Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China.
Nucleic Acids Res. 2021 Feb 22;49(3):1330-1344. doi: 10.1093/nar/gkaa865.
The occurrence of accidental mutations or deletions caused by genome editing with CRISPR/Cas9 system remains a critical unsolved problem of the technology. Blocking excess or prolonged Cas9 activity in cells is considered as one means of solving this problem. Here, we report the development of an inhibitory DNA aptamer against Cas9 by means of in vitro selection (systematic evolution of ligands by exponential enrichment) and subsequent screening with an in vitro cleavage assay. The inhibitory aptamer could bind to Cas9 at low nanomolar affinity and partially form a duplex with CRISPR RNA, contributing to its inhibitory activity. We also demonstrated that improving the inhibitory aptamer with locked nucleic acids efficiently suppressed Cas9-directed genome editing in cells and reduced off-target genome editing. The findings presented here might enable the development of safer and controllable genome editing for biomedical research and gene therapy.
CRISPR/Cas9 系统编辑基因组时发生的意外突变或缺失仍然是该技术尚未解决的关键问题。抑制细胞中 Cas9 过量或持续的活性被认为是解决此问题的一种方法。在这里，我们通过体外选择（指数富集的配体系统进化）和随后的体外切割试验筛选，开发了一种针对 Cas9 的抑制性 DNA 适体。该抑制性适体能以低纳摩尔亲和力与 Cas9 结合，并与 CRISPR RNA 部分形成双链体，从而发挥其抑制活性。我们还证明，用锁核酸改进抑制性适体可有效抑制细胞中 Cas9 指导的基因组编辑，并减少脱靶基因组编辑。本研究结果可能为生物医学研究和基因治疗中更安全和可控的基因组编辑的发展提供帮助。
