“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.”
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