Kuwasaki Yuto, Suzuki Kazushi, Yu Gaigai, Yamamoto Shota, Otabe Takahiro, Kakihara Yuki, Nishiwaki Michiru, Miyake Keita, Fushimi Keiji, Bekdash Ramsey, Shimizu Yoshihiro, Narikawa Rei, Nakajima Takahiro, Yazawa Masayuki, Sato Moritoshi
Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan.
Graduate School of Science and Technology, Shizuoka University, Shizuoka, Japan.
Nat Biotechnol. 2022 Nov;40(11):1672-1679. doi: 10.1038/s41587-022-01351-w. Epub 2022 Jun 13.
Red light penetrates deep into mammalian tissues and has low phototoxicity, but few optogenetic tools that use red light have been developed. Here we present MagRed, a red light-activatable photoswitch that consists of a red light-absorbing bacterial phytochrome incorporating a mammalian endogenous chromophore, biliverdin and a photo-state-specific binder that we developed using Affibody library selection. Red light illumination triggers the binding of the two components of MagRed and the assembly of split-proteins fused to them. Using MagRed, we developed a red light-activatable Cre recombinase, which enables light-activatable DNA recombination deep in mammalian tissues. We also created red light-inducible transcriptional regulators based on CRISPR-Cas9 that enable an up to 378-fold activation (average, 135-fold induction) of multiple endogenous target genes. MagRed will facilitate optogenetic applications deep in mammalian organisms in a variety of biological research areas.
红光能够深入穿透哺乳动物组织且光毒性较低，但目前开发的使用红光的光遗传学工具却很少。在此，我们展示了MagRed，一种红光可激活的光开关，它由一种吸收红光的细菌光敏色素组成，该光敏色素结合了哺乳动物内源性发色团胆绿素以及我们通过亲合体文库筛选开发的光态特异性结合剂。红光照射会触发MagRed两个组分的结合以及与之融合的分裂蛋白的组装。利用MagRed，我们开发了一种红光可激活的Cre重组酶，它能够在哺乳动物组织深处实现光激活的DNA重组。我们还基于CRISPR-Cas9创建了红光诱导型转录调节因子，能够对多个内源性靶基因实现高达378倍的激活（平均诱导倍数为135倍）。MagRed将促进在多种生物学研究领域对哺乳动物机体深处进行光遗传学应用。
