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.

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

“…The creation of specific DNA breaks can be obtained by the expression of nucleases such as zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs) and, more recently, the Cas9 protein associated with type II clustered regulatory interspaced short palindromic repeats (CRISPR) (Hsu et al ., 2014). CRISPR‐Cas9 relies on the presence of a 20‐nucleotide guide RNA (sgRNA) that targets specifically the Cas9 nuclease to the complementary genomic sequence (Jinek et al ., 2012). The sgRNA harbours the so‐called ‘protospacer adjacent motif’ (PAM), for which the consensus sequence, NGG, is adjacent to the 3′ end of the 20‐bp target.…”

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

“…The HNH nuclease domain of SpCas9 undergoes a substantial conformational rearrangement upon target binding 12–15 , which activates the RuvC nuclease for concerted cleavage of both strands of the DNA 12,16 . It was previously shown that the HNH domain stably docks in its active state with an on-target substrate, but becomes loosely trapped in a catalytically-inactive conformational checkpoint when bound to mismatched targets 10,12 .…”

Application of the CRISPR/Cas9 System to Drug Resistance in Breast Cancer

“…The sgRNA contains both a CRISPR RNA component (crRNA) as well as a trans‐activating crRNA, that binds to Cas9 and directs it to a specific sequence of interest via Watson–Crick base pairing ( Figure 1 ). 56 Multiplexed targeting by endonuclease Cas9 can be achieved at an unprecedented scale by employing a battery of short guide RNAs instead of a library of bulky proteins. The only criterion for defining the target is that it needs to be close to a PAM, DNA sequence, consisting of either NGG or NAG 57.…”