Supplementary Figure 9: In silico docking analysis reveals Acr mutations that improve CASANOVA performance.

(a) Light-dependent luciferase reporter cleavage mediated by different Acr–LOV hybrid mutants. HEK293T cells were co-transfected with vectors encoding (i) the indicated Acr–LOV hybrid variant, (ii) Cas9 and (iii) a luciferase reporter as well as a gRNA targeting the luciferase gene. Six hours post-transfection, cells were irradiated with pulsatile blue light for 48 h or kept in the dark as control before assessing the luciferase activity. Box plots show the median (center line) and first and third quartiles (box edges), 1.5× the interquartile range (whiskers) and individual data points (circles). n = 3 biologically independent samples (cell cultures). (b) T7 endonuclease assays and (c) corresponding quantification of light-mediated indel mutation of the human CCR5 locus. HEK293T cells were co-transfected with constructs expressing Cas9, the CCR5-locus-targeting gRNA and the indicated Acr–LOV hybrid variant and exposed to blue light for 70 h or kept in the dark as control. During transfection, the vector mass ratio of Acr–LOV:Cas9 construct was varied as indicated. Data represent a single experiment. n.d., not determined.