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Spatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here, to address this limitation, we present a screening platform which allows simultaneous measurements of the promoter strength, 5′, and 3′ processing efficiencies across a library of tRNA variants. This analysis reveals that the sequence determinants underlying these activities, while overlapping, are dissociable. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enables highly specific guide RNA production from a Pol-II promoter. When benchmarked against other reported systems this tRNA scaffold is superior to most alternatives, and is equivalent in function to an optimized version of the Csy4-based guide RNA release system. The results and methods described in this manuscript enable avenues of research both in genome engineering and basic tRNA biology.

Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression