Supplementary Figure 7: Cas9 inhibition can be modulated via mutations that affect docking of the LOV2 terminal helices.

(a) Light-dependent luciferase reporter cleavage mediated by different Acr–LOV hybrid mutants. HEK293T cells were co-transfected with plasmids 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 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 the Cas9, the CCR5-locus-targeting gRNA and the indicated Acr–LOV hybrid variant. During transfection, the vector mass ratio of Acr–LOV:Cas9 construct was varied as indicated. Subsequently, cells were exposed to blue light for 70 h or kept in the dark as control. The target locus was then PCR-amplified with primers flanking the estimated break point and the amplicon was denatured and re-annealed in a thermocycler to allow heteroduplex formation. Following digestion with T7 endonuclease, samples were analyzed on a 2% agarose gel (see Methods for details). (c) Data are means and dots indicate individual data points. n = 2 independent experiments.