CRISPR RNA guided surveillance complex Cascade system bound to a ssDNA-target

Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of RNA-guided adaptive immune systems that protect bacteria and archaea from viruses and plasmids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense).

In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kilodalton complex is called Cascade. We report the crystal structure of Cascade bound to a single-stranded DNA (ssDNA) target at a resolution of 3.03 angstroms. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This noncanonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding.

CRISPR RNA guided surveillance-complex Cascade system bound to a ssDNA target

CRISPR RNA-guided surveillance complex, Cascade, bound to a ssDNA target

The structure includes the strand of CRISPR RNA (red) and a short piece of the viral DNA (orange-yellow) after it has been unwound and recognized. The structure revealed a surprising but very logical structure for the RNA and DNA. The RNA is stretched open in a long spiral groove in Cascade, and the DNA binds side-by-side, instead of in the classical double helix.

Cas9 CRISPR associated protein 20 nt RNA guided endonuclease cleaves dsDNA target

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA.

REFERENCES

Crystal structure of a CRISPR RNA–guided surveillance complex bound to a ssDNA target. Sabin Mulepati, Annie Héroux, Scott Bailey. Published Online August 14 2014 Science 19 September 2014: Vol. 345 no. 6203 pp. 1479-1484 DOI: 10.1126/science.1256996

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