Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
Kanagawa Institute of Industrial Science and Technology, Kanagawa, Japan.
Methods Mol Biol. 2023;2577:229-240. doi: 10.1007/978-1-0716-2724-2_16.
The CRISPR-Cpf1 also known as Cas12a is an RNA-guided endonuclease similar to CRISPR-Cas9. Combining the CRISPR-Cpf1 with optogenetics technology, we have engineered photoactivatable Cpf1 (paCpf1) to precisely control the genome sequence in a spatiotemporal manner. We also identified spontaneously activated split Cpf1 and thereby developed a potent dCpf1 split activator, which has the potential to activate endogenous target genes. Here we describe a method for optogenetic endogenous genome editing using paCpf1 in mammalian cells. Furthermore, we show a method for endogenous gene activation using dCpf1 split activator in mammalian cells and mice.
CRISPR-Cpf1 也被称为 Cas12a，是一种类似于 CRISPR-Cas9 的 RNA 指导内切酶。我们将 CRISPR-Cpf1 与光遗传学技术相结合，设计了光激活的 Cpf1（paCpf1），以精确地在时空上控制基因组序列。我们还鉴定了自发激活的分裂 Cpf1，并由此开发了一种有效的 dCpf1 分裂激活剂，该激活剂有可能激活内源性靶基因。在这里，我们描述了一种在哺乳动物细胞中使用 paCpf1 进行光遗传学内源基因组编辑的方法。此外，我们还展示了一种在哺乳动物细胞和小鼠中使用 dCpf1 分裂激活剂进行内源基因激活的方法。
