Nature Communications, 11 June, 2019，DOI：http://dx.doi.org/10.1038/s41467-019-10507-3
Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d
Bo Zhang, Yangmiao Ye, Weiwei Ye, Vanja Per？ulija, Han Jiang, Yiyang Chen, Yu Li, Jing Chen, Jinying Lin, Siqi Wang, Qi Chen, Yu-San Han & Songying Ouyang
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.