Photoactivatable CRISPR-Cas9 for optogenetic genome editing

Abstract

We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications.

Light‐Patterned RNA Interference of 3D‐Cultured Human Embryonic Stem Cells

“…[15][16][17] The light-based optogenetic field has answered real biological questions [16] and developed tools for light-controlled genome editing and gene transfection. [18][19][20][21][22] However, these approaches rely largely on the visible light excitation sources and the construction of complex protein fusions via viral transfection. Other alternatives use photocaged small molecules and biopolymers for light-dependent gene regulation, [4,15,[23][24][25][26] which are limited by cellular delivery hurdles and the use of low tissue-penetrating UV-vis light.…”

dCas9: A Versatile Tool for Epigenome Editing

“…2.2C). Although the light-inducible CRY2-CIB1 pair works well to bring together dCas9 and effectors, the use of these partners was unsuccessful when applied to reassembly of split-(d)Cas9 (Nihongaki et al, 2015a). …”

“…2.2C). Such control has been achieved by using split-(d)Cas9 or split-(d)Cas9-effector proteins that are conditionally assembled into a functional DNAbinding complex in the presence of sgRNA (Wright et al, 2015), upon chemical induction (Zetsche et al, 2015b) or light induction (Nihongaki et al, 2015a). (d)Cas9, split in two individual domains, is assembled into a functional complex in the presence of full-length sgRNA.…”

“…Another strategy involves reassembly of splitdCas9 by photoinducible dimerization domains termed Magnets (pMag and nMag) (Fig. 2.2C) in response to exposure to blue light (Kawano et al, 2015;Nihongaki et al, 2015a).…”

Micromanagement with light

“…For example, Gardner and his colleague Laura Motta-Mena, a biochemist and cell biologist at the University of Texas Southwestern Medical Center in Dallas, have borrowed a light-activated transcription factor from bacteria to activate genes in a range of organisms 8 . At the University of Tokyo, meanwhile, chemist Moritoshi Sato and his colleagues have devised systems that use light to activate CRISPR-Cas9-based gene targeting to achieve high-precision control of gene editing or expression 9,10 . Optogenetic CRISPR tools such as these will be particularly useful for scientists who want to follow cell behaviour in entire organisms, Hahn says.…”