Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria

TLDR

CRISPR/Cas systems provide adaptive immunity in bacteria and archaea by silencing invading nucleic acids with ribonucleoprotein complexes guided by crRNAs. Cas9 cleaves DNA using its RuvC and HNH active sites to generate site‑specific nicks on opposite strands, guided by crRNA. The Cas9–crRNA complex acts as an RNA‑guided endonuclease that introduces double‑strand breaks at target sites, enabling programmable DNA cleavage and paving the way for engineered universal RNA‑guided nucleases.

Abstract

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9–crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9–crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.