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UC Berkeley Scientists Show Structure of CRISPR/Cas9 at Work

NEW YORK (GenomeWeb) – Scientists from the University of California, Berkeley have detailed how Cas9 changes the structure of its target DNA to position each strand for cleavage. 

Led by co-first authors Fuguo Jiang and David Taylor, and senior authors Jennifer Doudna and Eva Nogales, the scientists used X-ray crystallography and cryo-electron microscopy to solve several molecular structures of the Cas9 protein from Streptococcus pyogenes bound to both the guide RNA and target DNA, a formation that is known as an R-loop complex and is essential for the enzyme to cleave the target DNA. The scientists published their report today in Science.

The structures help explain how Cas9 holds unwound double-stranded DNA, which enables the formation of an RNA-DNA hybrid, without ATP-dependent helicase activity. "DNA binding at a sequence complementary to the 20-nt guide RNA segment in the Cas9–RNA complex induces protein structural rearrangements that accommodate both the RNA–DNA helix and the displaced non-target DNA strand," the authors wrote. "Those protein–nucleic acid interactions in turn direct the non-target DNA strand into the RuvC domain active site, favoring local conformational changes that position the HNH domain active site near the scissile phosphate of the target DNA strand." The protein induces a 30 degree bend in the helix that stabilizes the R-loop and allows the nuclease domains to cleave the double-stranded DNA.

The study clarifies the structure of Cas9 in action. More detailed knowledge of the Cas9-gRNA-DNA complex could lead to engineered variants of Cas9 enzymes that increase efficiency or decrease off-target effects. In recent months, for example, Feng Zhang of the Massachusetts Institute of Technology and Keith Joung of Massachusetts General Hospital and Harvard University have created new, more specific Cas9 variants based on hypotheses about how the enzyme functions.