UC San Diego

The discovery of CRISPR-Cas prokaryotic and archaeal immune systems has spurred the development of revolutionary precision molecular tools. While CRISPR DNA endonucleases have been rigorously characterized, novel RNA-targeting CRISPR systems are being explored as new discoveries rapidly emerge. Since their discovery, RNA-targeting CRISPR systems have quickly been applied for programmable transcript targeting, RNA therapeutics, and pathogen detection. Upon discovery, the Cas13d subtype was the smallest and most efficient RNA-targeting CRISPR system discovered. However, like all discovered Cas13 enzymes, Cas13d ribonucleases exhibit target-dependent collateral cleavage activity that limits the applicability of this technology. More recently, type III-E CRISPR-Cas effectors were characterized and exhibit significant RNA knockdown without collateral cleavage activity. Unlike Cas13 systems, type III-E effectors are very large and efficiencies can vary dramatically. In this body of work, we characterize and harness the collateral cleavage activity of Cas13d and subsequently establish a method to engineer and develop practical type III-E effectors for therapeutic applications.