The CRISPR/Cas9 bacterial genomic editing system identifies and cleaves complementary target sequences in foreign DNA. CRISPR (clustered regularly interspaced short palindromic repeats)–associated (Cas) protein Cas9 begins its work by RNA-guided DNA unwinding to form an RNA-DNA hybrid and displacing a DNA strand inside the protein. Upon binding, Cas9 reorganizes into an R-loop complex that is necessary for it to perform its function. A recent article published in Science describes work done to uncover the structural basis of Cas9’s function.
Jennifer Doudna (L) and Eva Nogales (R), both Howard Hughes Medical Investigators, UC Berkeley professors, and faculty scientists in the Molecular Biophysics & Integrated Bioimaging (MBIB) Division, led the research team. Damon Runyon Fellows and co-first authors of the article, Fuguo Jiang and David Taylor, determined the structure of this active formation of target-bound Cas9 using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). The data show how the non-target DNA strand is threaded through a side tunnel placing it in the right spot for nuclease cleavage. This action in turn moves another endonuclease domain into its proper position for concerted cutting of the target DNA strand. Structural and biochemical evidence indicates that Cas9 interacts with both ends of the open DNA helix, conferring a 30 degree bend, thereby creating the distortion necessary to stabilize the crucial R-loop complex. These findings explain how Cas9, an individual enzyme able to bring about R-loop formation, achieves this structural change leading to accurate, precise, and programmable DNA cleavage. Knowing how this protein functions provides insight into how it may work while editing genomes.