Optical Control of CRISPR/Cas9 Gene Editing

Abstract

The CRISPR/Cas9 system has emerged as an important tool in biomedical research for a wide range of applications, with significant potential for genome engineering and gene therapy. In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid. Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function.

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

“…These variants, including an intein-inactivated Cas9 system (Davis et al, 2015) and a small molecule-dimerized split Cas9 system (Zetsche et al, 2015b), have been shown to substantially improve genome editing specificity in mammalian cells compared with wild-type Cas9 by carefully controlling the temporal window within which active Cas9 is generated so that less active Cas9 is present after modification of the on-target loci is complete (Figure 2g,h). Similar systems, such as light-activated Cas9 variants (Nihongaki et al, 2015a;Hemphill et al, 2015;Jain et al, 2016), split Cas9 variants (Truong et al, 2015;Wright et al, 2015), small-molecule induction of Cas9 (Dow et al, 2015), and an engineered allosterically regulated Cas9 (Oakes et al, 2016) could also be used to reduce off-target genome editing following these same principles.…”

dCas9: A Versatile Tool for Epigenome Editing

“…In both cases, after intein transsplicing is induced full-length functional Cas9 is obtained (Davis et al, 2015;Truong et al, 2015). An other, less common approach, is to use Cas9 inactive due to caging of lysine residues necessary for Cas9 function (Hemphill et al, 2015). Exposure to UV light removes the caging group (Riggsbee and Deiters, 2010) and recovers an active Cas9 (Hemphill et al, 2015).…”

“…An other, less common approach, is to use Cas9 inactive due to caging of lysine residues necessary for Cas9 function (Hemphill et al, 2015). Exposure to UV light removes the caging group (Riggsbee and Deiters, 2010) and recovers an active Cas9 (Hemphill et al, 2015).…”

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.…”