LAUSR

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3′-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.Cas13d is a class 2 type VI-D CRISPR-Cas RNA-guided RNase. Here the authors present the high-resolution crystal structure of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex and by combining structural, mutational and biochemical studies provide mechanistic insights into the CRISPR-Cas13d system.