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.

中文翻译：

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用于光诱导基因编辑和转录的单链光开关CRISPR-Cas9架构

CRISPR-Cas9衍生蛋白的光学控制可用于将基因编辑或转录调控限制在所需的时间和位置。用光不可笼罩的非天然氨基酸或通过使用光诱导的蛋白质相互作用从两个多肽重构Cas9介导的功能已经实现了对Cas9功能的光学控制。但是，这些方法仅应用于一种Cas9物种，尚未用于对两个基因的不同扰动进行光学控制。在这里，我们使用可光解离的二聚体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结合裂隙在基线被遮挡，并在照明时打开。此设计成功控制了Cas9的不同物种和功能变体，介导的转录激活比以前的光遗传学方法更强大，并且能够在同一细胞中光诱导一个基因的转录并编辑另一个基因。因此，单链可光转换体系结构提供了控制各种Cas9介导的功能的通用方法。