Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage

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Science  19 Feb 2016:
Vol. 351, Issue 6275, pp. 867-871
DOI: 10.1126/science.aad8282

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CRISPR Cas9 molecular scissors

The CRISPR-associated (Cas) protein Cas9 is a molecular scissor for cutting DNA. The first step in the cutting reaction is the RNA-guided unwinding of the DNA double helix. Jiang et al. determined the structures of Cas9 bound to DNA unwound by the targeting RNA (see the Perspective by Chen and Bailey). Cas9 bends the DNA to allow guide RNA infiltration into the double helix. The two separated DNA strands, one bound to RNA, are subsequently positioned in the dual active sites of the protein for cutting.

Science, this issue p. 867; see also p. 811


Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)–associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.

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