Department of Bioengineering, Stanford University , Stanford, California, United States.
Department of Neurobiology, Stanford University , Stanford, California, United States.
ACS Chem Biol. 2018 Feb 16;13(2):443-448. doi: 10.1021/acschembio.7b00603. Epub 2017 Sep 29.
Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.
光学控制 CRISPR-Cas9 衍生蛋白将有助于将基因编辑或转录调控限制在特定的时间和地点。通过光解笼蔽非天然氨基酸或利用光诱导的蛋白质相互作用来重新组装 Cas9 介导的功能，已经实现了 Cas9 功能的光学控制，这来自两个多肽。然而，这些方法仅适用于一种 Cas9 物种，并且尚未用于在两个基因上对不同扰动进行光学控制。在这里，我们使用光解离二聚荧光蛋白结构域来工程化单链光可切换 Cas9（ps-Cas9）蛋白，其中 DNA 结合裂隙在基线处被阻断，在光照下打开。该设计成功地控制了 Cas9 的不同物种和功能变体，比以前的光遗传学方法更有效地介导转录激活，并能够在同一细胞中诱导一个基因的转录和另一个基因的编辑。因此，单链光可切换结构提供了一种通用方法来控制各种 Cas9 介导的功能。
