User:Wally Novak/Sandbox Brown

Cas9 is a large multifunctional protein that plays a central role in the CRISPR-Cas adaptive defense mechanism found in a vast amount of bacteria and archaea ^[1]. It accomplishes this through the use of antisense RNAs which serve as signatures from past viral invasions ^[2]. The adaptive immunity occurs in three stages: insertion of invading DNA into CRISPR locus, transcription of precursor crRNA from CRISPR locus that will be used to generate crRNA that matches its target sequence for 20 nucleotides, and crRNA-directed cleavage of foreign nucleic acids by cas9. PAM (protospacer adjacent motif) sequences must be present adjacent to the crRNA-targeted sequence to be cleaved ^[1]. In addition to the crRNA, cas9 incoporates another RNA chain that serves to anchor the crRNA to the protein. This tracrRNA is partially complimentary to a piece of the crRNA and interacts with an arginine-rich alpha helix to anchor both pieces of RNA to cas 9 ^[3]. Just in the last few years, this defensive mechanism and the cas9 protein has been used to develop genome engineering applications. TracrRNA:crRNA has been replaced by an engineered single guide RNA (sgRNA) that maintains the two main features of the RNA: the complementary 20-nucleotide long sequence at the 5' end and the double-stranded anchor at the 3' end to bind to cas9 ^[1]. The programmable cas9 protein is then used to create double-stranded breaks in genomic DNA, at which points the genetic sequence could then be altered.

Overall Structure

The cas9 protein complex has a seahorse shaped structure that is composed of 11 cas subunits. Cascade (CRISPR-associated complex for antiviral defense) is from the type-I CRISPR-cas system, and the crystal structure of this surveillance complex gives insight into the overall structure. (IMAGE) The body is comprised of six subunits (Cas7.1-7.6) wrapped around the crRNA in a helical filament with a dimer of Cse2 in the center ^[4]. The head of the Cas7 body is capped by Cas6e and the 3' end of the crRNA while the 5' end and Cas5 cap the tail. The N-terminal end of Cse1 is also at the tail and the C-terminal end contains a bundle of 4 helices that contact Cse2.2. The Cse2 dimer, Cas7 filament, and four-helix bundle of Cse1 form a groove immediately next to guide region of the crRNA. This is where the ssDNA target fits into the complex ^[4].

Binding Interactions with Target dsDNA

The groove in which the ssDNA target fits mentioned above is not formed until cas9 undergoes a conformational change upon association with a target dsDNA. The arginine-rich alpha helix to which tracrRNA binds serves as a hinge between the structural lobes of the overall structure. The conformational change is thought to take part in the R-loop formation that unwinds the target dsDNA and allows for interactions between crRNA and its complementary section ^[1].

The PAM sequence has been shown to be critical to inducing DNA binding, as Cas9 is unable to recognize even fully complementary sequences without it ^[1]. Upon formation of the substrate-protein complex, the nuclease and helical recognition lobes of Cas9 and the target ssDNA form a four-way junction straddling the arginine-rich alpha helix mentioned previously ^[5]. Nucleotides on either side of the PAM containing region (-1 to -8 on the target strand and +1 to +8 on the target strand) are base paired, and strand separation occurs only at the first base pair on the target strand (+1). The kink formed from the strand separation places the PAM sequence in a positively-charged groove known as the PAM-interacting domain ^[5].