General information on Cas9 Protein
Cas9 protein stands for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated protein 9. This protein is essential for the immunological defense of certain bacteria against DNA viruses. It’s a RNA-guided DNA endonuclease enzyme associated with the CRISPR adaptive immunity system. Cas9 protein has gained attention for its ability to be used as a genome engineering tool to induce site-directed double-strand breaks in DNA. These breaks can result in gene inactivation as well as the introduction of heterologous genes via non-homologous end joining and homologous recombination respectively. Native Cas9 requires a guide RNA composed of two disparate RNAs that associate – the CRISPR RNA (also known as crRNA), and the trans-activating crRNA (also known as tracrRNA).
One of the bacterias, S. pyogenes uses Cas9 to memorize, interrogate and cleave foreign DNA. In order to perform the interrogation, Cas9 unwinds foreign DNA and verifies for potential complementary sites to the guide RNA. Cas9 protein doesn’t cleave the invading DNA, unless the DNA substrate is complementary to guide RNA.
There are also versions of Cas9 protein that bind but do not cleave cognate DNA. This type of protein can be particularly useful for the location of transcriptional activator or repressors to specific DNA sequences. Native Cas9 requires a guide RNA composed of two disparate RNAs that associate – the CRISPR RNA (crRNA), and the trans-activating crRNA (tracrRNA). Cas9 targeting has been simplified through the engineering of a chimeric single guide RNA (chiRNA).
Cas9 protein features a bi-lobed architecture with the guide RNA nestled between the two. The lobes are linked through a single bridge helix. The multi-domain nuclease lobe has two nuclease domains, the RuvC responsible for the cleavage of the non-target DNA strand, and the HNH nuclease domain which cleaves the target strand of DNA. There areat least three crystal structures have been studied and published for this protein.