Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo, Japan.
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Methods Mol Biol. 2021;2312:225-233. doi: 10.1007/978-1-0716-1441-9_13.
The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.
CRISPR-Cas9 系统提供了简单的靶向基因组操作。我们将 CRISPR-Cas9 与光遗传学技术相结合，设计了光激活 Cas9，以精确地在时空上控制基因组序列。本文提供了使用光激活 Cas9 进行光遗传学基因组编辑实验的详细方案，包括向导 RNA 载体的生成、光介导的 Cas9 激活以及哺乳动物细胞中基因组编辑效率的定量。
