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A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs** |
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| Authors: | Dr Ying-Jie Sun Wen-Da Chen Dr Ji Liu Jun-Jin Li Yu Zhang Wei-Qi Cai Prof Li Liu Xin-Jing Tang Jian Hou Prof Ming Wang Prof Liang Cheng |
| Institution: | 1. Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Laboratory of Molecular Recognition and Function, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190 China;2. Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Laboratory of Molecular Recognition and Function, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190 China
University of Chinese Academy of Sciences, Beijing, 100049 China;3. BNLMS, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190 China;4. State Key Laboratory of Agrobiotechnology and College of Biological Science, China Agricultural University, Beijing, 100193 China;5. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191 China;6. BNLMS, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190 China University of Chinese Academy of Sciences, Beijing, 100049 China |
| Abstract: | The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches for precise control of genome editing and regulatory events are still desirable. Here, we report the spatiotemporal and efficient control of CRISPR/Cas9- and Cas12a-mediated editing with conformationally restricted guide RNAs (gRNAs). This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to the heavily modified linear gRNAs that often require extensive screening and time-consuming optimization. This tactic could direct the precise cleavage of the genes encoding green fluorescent protein (GFP) and the vascular endothelial growth factor A (VEGFA) protein within a predefined cutting region without notable editing leakage in live cells. We also achieved light-mediated myostatin (MSTN) gene editing in embryos, wherein a new bow-knot-type gRNA was constructed with excellent OFF/ON switch efficiency. Overall, our work provides a significant new strategy in CRISPR/Cas editing with modified circular gRNAs to precisely manipulate where and when genes are edited. |
| Keywords: | CRISPR/Cas Circular gRNA Gene Editing Photo-Modulation Spatiotemporal Control |