691. A Light-Inducible CRISPR/Cas9 System for Control of Endogenous Gene Activation

Abstract

Optogenetic systems enable precise spatiotemporal control of gene expression and cellular behavior that is easily reversible and repeatable. Optogenetic technologies that facilitate dynamic control of endogenous genes would facilitate the systematic analysis of the relationship of gene expression patterns and cell differentiation or disease treatment. We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of gene-specific guide RNAs (gRNAs) and blue light. This system is based on the plant proteins CRY2 and CIB1 from Arabidopsis thaliana that heterodimerize in response to blue light. The full-length CRY2 was fused to the N-terminus of the transcriptional activator VP64 (CRY2FL-VP64), and an N-terminal fragment of CIB1 was fused to the N- and C-terminus of the catalytically inactive form of Cas9 (CIBN-dCas9-CIBN). When these fusion proteins are expressed with a gRNA, CIBN-dCas9-CIBN localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins.Light-dependent activation of the IL1RN, HBG, or ASCL1 genes was achieved by delivery of the LACE system and four gene-specific gRNAs per promoter region. Illuminated cells in which IL1RN or HBG1/2 was targeted demonstrated significantly greater mRNA levels in the light compared to the dark (p<0.0001 and 0.005, respectively), as well as equivalent activation levels to dCas9-VP64 (p=0.17 and 0.35, respectively). Significant light-dependent activation was also observed when the ASCL1 locus was targeted with the LACE system (p<0.0001). However, in this case mRNA levels were not activated to the same extent as cells that received dCas9-VP64 and the same four ASCL1-targeting gRNAs. In all instances, transfected cells incubated in the dark maintained levels of the targeted gene that did not significantly differ from mock-transfected cells. Importantly, the fusion of CIBN to both N- and C-termini of dCas9 yielded 10- to 100-fold greater gene activation than when CIBN was fused to only one terminus.Endogenous gene expression could be controlled in a reversible and repeatable fashion by modulating the duration of blue light exposure. Spatial patterning of gene expression was also achieved using an eGFP reporter. Cells illuminated though a photomask containing slits of varying width resulted in a corresponding pattern of eGFP-expressing cells. This versatile LACE system is a valuable tool for studying dynamic gene expression patterns in synthetic biology, basic science, and tissue engineering. View Large Image | Download PowerPoint Slide