CAS9 is a restrictive enzyme used to cut targeted pieces of DNA.

CAS9 is
introduced into a cell, binds to PAM motif, and cleaves both DNA strands when the
DNA and guide RNA match.

PAM is a three nucleotide sequence motif that is abundant within a

genome. It is where CAS9 binds once it recognizes it, and CAS9 will start
unwinding the DNA double helix from that location.

The role of guide DNA is to nd its matching segment in the DNA at the
location where the CAS9 binds. Once it does, it will bind to the DNA
through complementary base pairing.

The pairing of DNA and RNA will trigger CAS9 to switch its three
dimensional form and activate its nuclease activity. The CAS9 cuts the
DNA strands at a place up in the DNA from PAM.
Although cells contain many DNA repair enzymes, the process of
repairing DNA is very vulnerable to mistakes that can lead to single
nucleotide mutations or other mutations. This may accidentally
inactivate a gene, so it is risky to break DNA

CRISPR is found naturally in bacteria. They use the ability to manipulate

their cells and DNA as an evolutionary defense mechanism against
bacteriophages.

CAS9 can be catalytically disabled so that it can’t cut double stranded

DNA. This inhibited form of CAS9 can still be used to bind to a target site
and bring an enzyme, like cytidine deanimase, to convert C bases to Ts
like a base editor.

CRISPR-CAS9 can be utilized to make two cuts in DNA, anking the

mutation. With that section of the gene removed, the gene can be
transcribed and translated to produce a protein that lacks the internal
middle section. Though, the protein can still go end to end and have
partial function so the muscle can partially restore function.