fachgruppenaerzte-1
Startseite

Paper of the Month

CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9

Dugar G, Leenay RT, Eisenbart SK, Bischler T, Aul BU, Beisel CL, Sharma CM
Molecular Cell, 2018, 69(5):893-905 

Vollständiger Artikel: https://www.ncbi.nlm.nih.gov/pubmed/29499139externer Link

DugarFoto: Dr. Gaurav Dugar

Kommentar: CRISPR-Cas9-Nukleasen schneiden typischerweise doppelsträngige DNA. In unserem Paper zeigen wir, dass die Cas9-Nuklease des Typ II CRISPR-Cas Systems des Lebensmittelkeims Campylobacter jejuni auch endogene RNAs binden und schneiden kann. Die Bindung und Spaltung von RNAs durch C. jejuni Cas9 (CjCas9) erfolgt hierbei durch imperfekte Komplementarität mit nativen crRNA-Guides. Zudem konnten wir die RNA-Bindung und Spaltung durch C. jejuni Cas9 in vitro gegen spezifische RNAs programmieren, was neue Anwendungsmöglichkeiten von CjCas9 bezüglich RNA-Targeting eröffnet.

Abstract: Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.

Letztauthor/Corresponding author:
 Prof. Dr. Cynthia M. Sharma
 Molekulare Infektionsbiologie II
 Institut für Molekulare Infektionsbiologie(IMIB)
 Universität Würzburg
 Josef-Schneider-Straße 2 / Bau D15
 97080 Würzburg
 Tel: +49-931/31-82560
 E-Mail: cynthia.sharma@uni-wuerzburg.de