Fig. 1: Engineering and characterization of CASANOVA, an anti-CRISPR protein dependent on blue light. | Nature Methods

Fig. 1: Engineering and characterization of CASANOVA, an anti-CRISPR protein dependent on blue light.

From: Engineered anti-CRISPR proteins for optogenetic control of CRISPR–Cas9

Fig. 1

a, Schematic of CASANOVA function. b, Structural analysis of AcrIIA4. Top: solvent accessibility (access.) in the Cas9-bound and unbound states. Center: contact map showing residues in close proximity (≤7-Å distance). Light red bars indicate Cas9-binding segments. Lower right: surface representation of AcrIIA4 (PDB 5VW1) with Cas9-interacting segments shown in light red. L, loop. c, Light control of luciferase reporter cleavage. HEK293T cells expressing Cas9, a luciferase reporter (Rep), a reporter-targeting gRNA and the indicated Acr–LOV variant (Supplementary Fig. 2) were irradiated with blue light for 48 h or kept in the dark and then subjected to a luciferase assay. n = 9 biologically independent samples (cell cultures). Acr-2A-LOV, control construct coexpressing AcrIIA4 and LOV2 via a P2A sequence. df, Light-mediated indel mutation of human CCR5 (d), CFTR (e) and EMX1 (f) loci. Cells expressing the indicated components were illuminated for 70 h or kept in the dark and then subjected to a T7 endonuclease assay. The vector mass ratio of the Acr:Cas9 construct is indicated. WT, wild-type; CN, CASANOVA. *P < 0.05, **P < 0.01, ***P< 0.001, two-sided Student’s t-test (exact P values are stated in the Methods section). d, n = 5 independent transfection and 3 independent transduction experiments. e,f, n = 3 independent experiments. cf, Box plots show the median (center line), first and third quartiles (box edges), 1.5× the interquartile range (whiskers) and individual data points (blue and gray symbols).

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