CRISPR Switches: Non Sequence-Constrained RNA-Sensing Grnas

We all like CRISPR, for its uses in genome engineering or for the panoply of engineered Cas proteins that have since transformed CRISPR into an RNA-guided platform with diverse uses. The presence of an RNA moiety in CRISPR-Cas systems offers the opportunity to link the field of CRISPR with that of RNA engineering, which uses secondary structure modelling to create artificial RNA sequences that can carry out logical operations. We have designed engineered Cas12a gRNAs that are rendered constitutively inactive by the addition of artificial structural elements, but can be switched back on upon the addition of a second trigger RNA. Making use of a cell-free transcription-translation system to characterise the engineered gRNAs, we were able to explore a diversity of designs for these CRISPR switches, shedding light on the importance and optimal design of the various structural elements that compose them. This permitted the creation of RNA-sensing CRISPR gRNAs that can be activated by artificial or natural RNA sequences. The switches are not sequence-constrained by the Cas12a handle or by the guide sequence, so could theoretically be used to detect any sequence without additional control elements. They can be used to control the activity of Cas proteins in vitro and in vivo, providing an additional layer of control and permitting the activation of Cas proteins upon the expression of an exogeneous or endogenous RNA.