Development of aptamer-based inhibitors for CRISPR/Cas system |
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| Authors: | Jing Zhao Rika Inomata Yoshio Kato Makoto Miyagishi |
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| Affiliation: | 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;Program in Life Science Innovation, School of Integrative and Global Majors, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan |
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| Abstract: | 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. |
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