Department of Chemistry, Case Western Reserve University, 2080 Adelbert Road, Cleveland, Ohio 44106, United States.
J Am Chem Soc. 2023 Mar 8;145(9):5561-5569. doi: 10.1021/jacs.3c01087. Epub 2023 Feb 22.
The CRISPR/Cas13b system has been demonstrated as a robust tool for versatile RNA studies and relevant applications. New strategies enabling precise control of Cas13b/dCas13b activities and minimal interference with native RNA activities will further facilitate the understanding and regulation of RNA functions. Here, we engineered a split Cas13b system that can be conditionally activated and deactivated under the induction of abscisic acid (ABA), which achieved the downregulation of endogenous RNAs in dosage- and time-dependent manners. Furthermore, an ABA inducible split dCas13b system was generated to achieve temporally controlled deposition of mA at specific sites on cellular RNAs through conditional assembly and disassembly of split dCas13b fusion proteins. We also showed that the activities of split Cas13b/dCas13b systems can be modulated by light via using a photoactivatable ABA derivative. Overall, these split Cas13b/dCas13b platforms expand the existing repertoire of the CRISPR and RNA regulation toolkit to achieve targeted manipulation of RNAs in native cellular environments with minimal functional disruption to these endogenous RNAs.
CRISPR/Cas13b 系统已被证明是一种强大的 RNA 研究和相关应用工具。新的策略能够精确控制 Cas13b/dCas13b 的活性，同时最小化对天然 RNA 活性的干扰，这将进一步促进对 RNA 功能的理解和调控。在这里，我们构建了一个可在脱落酸（ABA）诱导下条件激活和失活的分裂 Cas13b 系统，该系统能够以剂量和时间依赖的方式下调内源性 RNA。此外，我们还构建了一个 ABA 诱导的分裂 dCas13b 系统，通过分裂 dCas13b 融合蛋白的条件组装和拆卸，实现在细胞 RNA 上特定位置的 mA 进行时间控制沉积。我们还表明，通过使用光激活的 ABA 衍生物，可以调节分裂 Cas13b/dCas13b 系统的活性。总的来说，这些分裂 Cas13b/dCas13b 平台扩展了现有的 CRISPR 和 RNA 调控工具包，以实现对天然细胞环境中 RNA 的靶向操作，同时对这些内源性 RNA 的功能干扰最小。
