Extended Data Fig. 6: Grafting cpLOV2 into AcrIIA4 to create a light-switchable anti-Cas9 inhibitor (LiCASINO).
From: Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering
a, The 3D structure of the spCas9-sgRNA-AcrIIA4 complex. Sphere, cpLOV2 insertion sites. b, The ___domain organization of AcrIIA4-cpLOV2 hybrids. V5 showed the most prominent light-dependent activity. Linkers (GGSG or GGSGG) used to flank the grafted cpLOV2 into the host protein were indicated. c, Light-inducible Cas9-inhibitory activity of LiCASINO. (left) Cartoon depicting the design of the screening assay. HEK293T cells were co-transfected with dCas9-VPR, the indicated AcrIIA4-cpLOV2 variants, and an sgRNA targeted to a synthetic promoter upstream of GFP. In the dark, LiCASINO tightly binds dCas9 to prevent its binding to the target genomic locus. Upon light irradiation, cpLOV2 alters the conformation of AcrIIA4 to diminish its dCas9-inhibitory activity, thereby restoring the function of dCas9-VPR to turn on GFP expression. (right) Quantification of light-inducible changes in GFP expression (n = 3 independent biological replicates; mean ± s.e.m.). VPR, a synthetic transcriptional coactivator made of VP64, p65 and Rta. P values are determined by unpaired two-tailed Student’s t-test. d, T7E1 assay to confirm photo-controllable genome editing at an endogenous genomic locus. HEK293T cells were co-transfected with Cas9, LiCASINO, and an sgRNA designed to disrupt the endogenous CCR5 gene. e, Immunoblot analysis of the effect of light on the interaction between FLAG-Cas9 and LiCASINO-mCh2. HEK293T cells were co-transfected with both constructs. FLAG-Cas9 was immobilized on anti-FLAG beads as the bait to pulldown the indicated AcrIIA4 variants. WT AcrIIA4 was used as a positive control and GAPDH was used as loading control.
