The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is the latest RNA-guided, endonuclease tool in genome editing which allows for very specific genomic disruption and replacement.
Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme associated with the CRISPR adaptive immunity system in S. pyogenes, among other bacteria. S. pyogenes utilises Cas9 to memorise and later interrogate and cleave foreign DNA such as invading bacteriophage DNA or plasmid DNA.
Cas9 Nuclease protein forms a very stable ribonucleoprotein (RNP) complex with the guide RNA (sg RNA) of the CRISPR/Cas9 system and unwinds the genomic DNA duplex and cleaves both strands upon recognition of the target sequence by the sg RNA. The resulting doublestranded break gets repaired by the non-homologous end joining (NHEJ) pathway, leading to a disruption in the open reading frame of the targeted gene.