Site-Specific and Enzymatic Cross-Linking of sgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing.
Site-Specific and Enzymatic Cross-Linking of sgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing.
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DOI：
10.1021/jacs.1c12166
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发表时间：
2022-03-16
影响因子：
15
通讯作者：
Devaraj, Neal K.
中科院分区：
文献类型：
--
作者：
Zhang, Dongyang;Liu, Luping;Jin, Shuaijiang;Tota, Ember;Li, Zijie;Piao, Xijun;Zhang, Xuan;Fu, Xiang-Dong;Devaraj, Neal K.
化学交联使得能够快速鉴定RNA-蛋白质和RNA-核酸分子间和分子内相互作用。然而，不存在位点特异性和共价交联RNA内的两个用户定义的位点的方法。在这里，我们开发了RNA-CLAMP，它能够在RNA内对两个选定的鸟嘌呤残基进行位点特异性和酶促交联（夹紧）。分子内钳位可以破坏正常的RNA功能，而随后的交联剂的光裂解恢复活性。我们使用RNA-CLAMP通过可光裂解的交联剂在CRISPR-Cas9基因编辑系统的单向导RNA（sgRNA）内夹持两个茎环，完全抑制编辑。可见光照射切割交联剂并以高时空分辨率恢复基因编辑。设计两个响应于不同波长的光的光可裂解接头允许在哺乳动物细胞中基因编辑的多重光活化。这种光激活的CRISPR-Cas9基因编辑平台受益于不可检测的背景活性，提供了激活波长的选择，并具有多路复用能力。
Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the crosslinker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting editing. Visible light irradiation cleaved the crosslinker and restored gene editing with high spatiotemporal resolution. Design of two photocleavable linkers responsive to different wavelengths of light allowed multiplexed photo-activation of gene editing in mammalian cells. This photo-activated CRISPR-Cas9 gene editing platform benefits from undetectable background activity, provides a choice of activation wavelengths, and has multiplexing capabilities.
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