Figure 1.

Light-induced CRISPR-Cas-mediated target DNA cleavage. (a) Schematic representation of photocaged gRNAs mechanism of action. (b) Sequences and modification strategies of PC-crRNAs. AUGC are RNA bases, P is 6-nitropiperonyloxymethyl (NPOM)-caged T-DNA, asterisks denote phosphorothioate linkages, underlined are targeting sequences. (c) Kinetics of PC-crRNA light-mediated uncaging reaction monitored by reverse-phase high-performance liquid chromatography (N = 3, mean ± SD). (d) Biochemical activity assay of target DNA cleavage by noncaged (NC) or photocaged (PC) gRNAs with 365 nm light illumination (0–2 J/cm²). (e) Quantification of target DNA cleavage from gel images using Fiji ImageJ software.⁴⁴ Results are expressed as individual data points overlaid with the mean ± SD (N = 3); ***p < 0.001, ns–not significant (p > 0.05) versus NC-gRNA according to one-way ANOVA analysis combined with Tukey’s (Holm-Sidak) posthoc test. Data are fitted with B-spline function.