Recombinant dCas9 protein, His/FLAG Tag
dCas9 (deficient Cas9) protein is an endonuclease-inactive version of Cas9 (CRISPR associated protein 9). Cas9 is an RNA-guided DNA endonuclease associated with adaptive immunity system in Streptococcus pyogenes, among other bacteria. Cas9 can unwind foreign DNA and checking for sites complementary to the 20 basepair spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In this sense, the CRISPR-Cas9 mechanism has a number of parallels with the RNA interference (RNAi) mechanism in eukaryotes. dCas9 lacks of the endonuclease activity, so that it bind but do not cleave cognate DNA, which can be used to localize transcriptional activator or repressors to specific DNA sequences in order to control transcriptional activation and repression. While native Cas9 requires a guide RNA composed of two disparate RNAs that associate to make the guide – the CRISPR RNA (crRNA), and the trans-activating RNA (tracrRNA), dCas9 targeting has been simplified through the engineering of a chimeric single guide RNA (chiRNA).