Giedrius Gasiūnas1, Rodolphe Barrangou2, Philippe Horvath3, Virginijus Šikšnys4
1Institute of Biotechnology, Vilnius University, LT-02241 Vilnius, Lithuania;
2bDuPont Nutrition and Health, Madison, WI 53716; and
3DuPont Nutrition and Health, F-86220 Dangé-Saint-Romain, France
4aInstitute of Biotechnology, Vilnius University, LT-02241 Vilnius, Lithuania;
Tóm tắt
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.
