If I understand correctly, the steps of gene editing with CRISPR-Cas9 are roughly as follows
- Cas9 nuclease and guide RNA form a complex.
- Cas9+guide RNA complex scans genomic DNA and recognizes the sequences homologous to the guide RNA
- Induces a double strand break in DNA upstream of the PAM sequence (only breaks when the homologous sequence of the guide RNA is in the vicinity of the PAM).
- Genetic modification using the mechanism of DNA repair (NHEJ, HDR) after double-strand break.
If so, in the gene editing with CRISPR-Cas9 , gene editing is likely to continue as long as the Cas9 does not lose its activity, am I right?
- When does the Cas lose its activity?
- What was the logic of the experiment that identified the timing? / If the matter itself is unexplored, what kind of experiments could be done to find out the timing when the Cas lose its activity?
As this movie shows, if the gene editing occurs successfully, it will indeed introduce a mutation in the target gene.
However, even if it does,-this is just my guess from here on out- it is likely to remain complementary to the target gene, at least upstream of the guide RNA. However, even if the target gene is mutated, there is likely to remain complementary sequence to the guide RNA, at least upstream of the mutation. So, even if one successful gene edit is completed, I think it's possible that gene editing could happen again in the same location. Moreover, even if the targeted site is successfully edited, there is still the possibility of subsequent off-target editing elsewhere. That's why I think the tools exist to stop CAS.
A guide RNA binds to the target gene that has been bitten by CAS. The strand opposite the strand to which the guide RNA was bound has just been cleaved. Quoted from this video.
An idea for an experiment to detect when CAS goes quiet
If a single cell colony was created from a gene-edited population and there was genetic variation in the cells born from that colony, then I think that would mean that there would have been Cas9 activity even after the single cell colony was started in culture. Is this idea correct? Could there be a smarter experiment?