CRISPR-Cas9 trans-cleavage is hindered by a flanked R-loop, an elongated spacer, and an inactive HNH domain

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. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Analysis of structural models provides tentative mechanistic insights. Together, these findings illustrate that fine modulation of Cas9 function can be achieved. Prokaryotic Class 2 CRISPR-Cas nucleases like Cas9 exhibit collateral cleavage activity. Here, the authors show that this activity is supported by unflanked R-loops in the RNA 5′ side, spacers of canonical length of 20 nt, and through its HNH and RuvC domains.