Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system is a system that replaces the existing protein-based targeting (TALEN and Zinc Finger) method with RNA-Guided Endonuclease Technology which is currently the most commonly used for genome engineering and is a system that allows nuclease to find DNA target using short RNA
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) systems are defense mechanisms against external DNA found in bacteria. To date, three types of bacterial CRISPR systems have been identified. Of them, Type II system is CRISPR, which is now the basis of genome engineering technology.
This system consists of 1) guide RNA and 2) endonuclease (CRISPR associated (Cas) nuclease, Cas9). When these guide RNA and Cas9 are expressed in the cell, the genomic target sequence can be modified or permanently damaged.
The gDNA / Cas9 complex forms base-pairing with the target sequence of the genomic DNA complementary to the gRNA sequence. In order for Cas9 to successfully bind to the target sequence, the correct Protospacer Adjacent Motif (PAM) sequence must be located immediately following the genomic target sequence. If Cas9 binds to the target sequence, both strands of DNA are cut to produce Double Strand Break (DSB). Cas9 cuts the upstream 3-4 nucleotides of the PAM sequence. The DSB created in this way is repaired to one of two repair pathways: 1) Non-Homologous End Joining (NHEJ) DNA repair pathway 2) Homology Directed Repair (HDR) pathway.
The NHEJ repair pathway can induce frameshifts or premature stop codons at the DSB site and damage the open reading frame (ORF) of the target gene due to inserts / deletions (InDels). The HDR pathway requires a repair template to repair the DSB. HDR can accurately copy the repair template to cut the target sequence and then introduce the specific sequence into the target gene with the repair template.