CRISPR/Cas9, is an RNA-guided targeted genome editing tool – allows researchers to do gene knockout, knocking SNPs, insertions and deletions in cell lines and animals. The CRISPR/Cas9 genome editing system requires two components, Cas9, the endonuclease, and a guide RNA (sgRNA); sgRNA guides Cas9 to the location in the genome sequence specifically. With the protospacer-adjacent motif (PAM - the sequence NGG) present at the 3′ end, Cas9 will unwind the DNA duplex and cleave both strands upon recognition of a target sequence by the guide RNA. The CRISPR-Cas9 system consists of two key molecules that introduce a mutation into the DNA. These are: an enzyme called Cas9. This acts as a pair of ‘molecular scissors’ that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed. A piece of RNA called guide RNA (gRNA). This consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome. The guide RNA is designed to find and bind to a specific sequence in the DNA. The guide RNA has RNA bases that are complementary to those of the target DNA sequence in the genome. This means that, at least in theory, the guide RNA will only bind to the target sequence and no other regions of the genome. The Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut across both strands of the DNA. At this stage the cell recognizes that the DNA is damaged and tries to repair it. Scientists can use the DNA repair machinery to introduce changes to one or more genes in the genome of a cell of interest.
Assistance of Aqief Afzal
Research Assistant, ASRBC