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 TracrRNA:crRNA has been replaced by an engineered single guide RNA (sgRNA) that maintains the two main features of the RNA: the complementary 20-nucleotide long sequence at the 5' end and the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 contains two nuclease domains, HNH and RuvC

Active Site Structure

Structural highlights

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