User:Ben.lafrance/sandbox
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Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria. S. pyogenes utilizes Cas9 to interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for complementarity with the 20 base pair long spacer region of the guide RNA. If a DNA substrate is cognate 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.
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Apart from its original function in bacterial immunity, the Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double strand breaks in DNA. These breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in many laboratory model organisms. Alongside zinc finger nucleases and TALEN proteins, Cas9 is becoming a prominent tool in the field of genome editing.
Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide RNA.[1] Because the target specificity of Cas9 stems from the guide RNA:DNA complementarity and not modifications to the protein itself (like TALENs and Zinc-fingers), engineering Cas9 to target new DNA is quite easy.[2] The design flexibility coupled with versions of Cas9 that binds but does not cleave cognate DNA also has potential for turning genes on and off by localizing transcriptional activator or repressors to specific DNA sequences.[3][4] Further simplification was provided in a 2012 seminal paper that depicts the creation of a chimeric single guide RNA, rather than the original guideRNA comprising of two disparate RNAs that associate to make the guide- the CRISPR RNA (crRNA), and the trans-activating RNA (tracrRNA).[5] Scientists have suggested that Cas9-based gene drives may be capable of editing the genomes of entire populations of organisms.[6] Much like the revolution in molecular biology that accompanied the discovery of restriction enzymes in the 1970s, the “Cas9 toolbox” also holds great potential.