CRISPR-Cas molecular mechanism
The CRISPR-Cas system molecular mechanism is based on its components. In order for the system to function, there are two important conditions to meet:
-
The target sequence should be as unique as possible compared to the rest of the genome.
-
The target sequence should be immediately adjacent to the PAM sequence
These are the main steps (Figure 1):
-
The gRNA and the Cas protein form the Ribonucleoprotein (RNP) complex in the cytoplasm, and enter the nucleus with the help of a protein called Importin.
-
The RNP complex looks for the specific PAM sequence in the genome. Once it finds one, the gRNA will try to anneal to the target from the 3' end of the spacer gRNA. If after ca. 10 bases both sequences anneal correctly, the gRNA will continue in the same 3' to 5' direction like a zipper. It is, therefore, very important that the first 10 bases of the spacer sequence in the gRNA (also known as seed sequence) match the target DNA to permit the cleavage.
-
Then, the Cas protein will use its enzymatic activity to modify the target DNA. In the case of the most commonly used Cas9, it will produce a double-strand break at a given locus (normally around 3-4 nucleotides upstream of the PAM sequence), leaving blunt ends in both DNA strands.
Figure 1. CRISPR-Cas mechanism.
Once CRISPR-Cas has modified the target DNA, the repair pathways are triggered, being the non-homologous end joining (NHEJ) the most active one, which in the case of using CRISPR-Cas9 normally leads to the generation of knock-out cells.