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Abstract Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key determinants underlying this function. First, we show that unflanked R-loops in the RNA 5′ side favor trans-cleavage activity, which occur when targeting short double-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impair this collateral activity.
Introduction Click to copy section linkSection link copied! Engineered optogenetic systems represent a significant biotechnological advance to control gene expression and protein activity in vivo with high spatiotemporal precision. (1) In contrast to chemical inducers, light is not limited by diffusion and shows higher orthogonality in addition to being cost-effective.
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