Supplementary Figure 5: Photoactivatable genome editing with CASANOVA.
From: Engineered anti-CRISPR proteins for optogenetic control of CRISPR–Cas9
Light-mediated indel mutation of human CFTR locus (a,b; with two different gRNAs), mir-122 locus (c) and VEGFA locus (d). HEK293T cells were co-transduced with AAV vectors encoding CASANOVA, Cas9 and the indicated gRNA and 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). Representative gel images and corresponding quantifications of editing frequencies are shown. Wt, wild-type. 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 independent experiments. All statistics by two-sided Student’s t-test. (a) ***P = 5.83 × 10–5 (b) **P = 0.0039 (c) ***P = 2.85 × 10–5 (d) ***P = 2.01 × 10–5.
