title: A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
authors: Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
authors: Martin
authors: M
authors: Jinek
authors: Krzysztof
authors: K
authors: Chylinski
authors: Ines
authors: I
authors: Fonfara
authors: Michael
authors: M
authors: Hauer
authors: Jennifer A
authors: JA
authors: Doudna
authors: Emmanuelle
authors: E
authors: Charpentier
mesh_terms: D001435:Bacteriophages / Q000276:immunology*; D001483:Base Sequence; D053903:DNA Breaks, Double-Stranded; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000502:physiology
chemical_list: D012313:RNA; D015252:Deoxyribonucleases, Type II Site-Specific