Synthetic type III-E CRISPR-Cas effectors for programmable RNA-targeting

The recent discovery of the type III-E CRISPR-Cas effector class has reshaped our fundamental understanding of CRISPR-Cas evolution and classification. Type III-E effectors are composed of several Cas7-like domains and a single Cas11-like domain naturally fused together to create a single polypeptide capable of programmably targeting and degrading RNA. Here we identify a novel composition of a type III-E-like effector composed of Cas7-like and a Cas1-like domain, that can be engineered into an active chimeric RNA-targeting Cas effector and presents a new function of Cas1 in RNA-targeting. Furthermore, we demonstrate a unique modularity of type III-E effectors by methodically substituting domains between orthogonal type III-E proteins to engineer compact synthetic Cas effectors. We refine our methods to generate several compact effectors for programmable RNA-targeting in mammalian cells. Cas7-S represents a new understanding of type III-E architecture and modularity, and provides a platform for engineering genome engineering technologies from the blueprint of nature.