Extended Data Fig. 1: Optimization of a conditional gRNA.

14 conditional gRNA variants were tested for potential leakiness (activity in the absence of the trigger gRNA). We examined the ability of the conditional gRNA to activate, via SSA, a specific conditional reporter¹¹ (gRNA-spacer reporter, green) in the absence of the trigger gRNA. Each variant was tested with both Dpn-Cas9 and Actin-Cas9. Given that the target sequence (embedded in the scaffold) in the variant #5 unexpectedly acted as an active gRNA, we included a second reporter (gRNA-target reporter, red) to analyze this activity. From the variant #8 onwards we avoided this activity by inverting the orientation of the target sequence. Note that variant #6 is the least leaky as it shows little activity with Actin-Cas9 and almost no activity at all with Dpn-Cas9. Green/Red, immunohistochemistry for EGFP/mCherry. Gray, nc82 counterstain. See also Supplementary Text. n=10 brains. Scale bar = 50 μm.