Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 31186424.
- Also identified by DOI 10.1038/s41467-019-10507-3 and PMC identifier 6559982.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
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 Mg<sup>2+</sup> 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.
Medical subject headings
- Bacterial Proteins
- CRISPR-Associated Proteins
- Clustered Regularly Interspaced Short Palindromic Repeats
- Ribonucleases
- Ruminococcus