6
$\begingroup$

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?

My question

  • 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.

enter image description here
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?

$\endgroup$
5
  • 1
    $\begingroup$ Once the cut is made and DNA of interest is inserted will the guide RNA have the same template to bind to? $\endgroup$
    – Roni Saiba
    Nov 28 '20 at 7:18
  • $\begingroup$ @ Roni Saiba Thanks for your comment. Is your answer that the target gene in the template after editing is no longer able to bind to the guide RNA by mutation occurred on the template side? But even if mutation does occur, can the guide RNAs still bind to the target gene? The ability to bind may be weakened, but it may still be possible to bind. Otherwise, there would be no such thing as "Cas that can be deactivated by light" or "a peptide to stop Cas9". $\endgroup$ Nov 28 '20 at 10:31
  • $\begingroup$ Perhaps the more Chunked downed question might be to create an assay to compare "which method of stopping is more certain to stop the CAS". If I look for a paper on the means to stop CAS, perhaps I will find the answer to this question. Because such experiments would be necessary to clarify whether peptides or the light (Written in Japanese) can really stop CAS. $\endgroup$ Nov 28 '20 at 10:37
  • $\begingroup$ the deactivation mechanisms may also have been placed to stop off target activity after the job at the primary site is done. But I am in pure speculation territory now. I'll add an answer if I find enough material with references to convince myself of the mechanism. $\endgroup$
    – Roni Saiba
    Nov 28 '20 at 11:51
  • $\begingroup$ @ Roni Saiba Thank you for your help. Maybe the extent of the subsequent CAS rampage may vary depending on what part of the target gene the mutation enters. If I understand it correctly, the way the mutation occurs is a matter of probability. So I think this variation in probability should be taken into account in order to "identify when the CAS will become docile." $\endgroup$ Nov 28 '20 at 11:58
2
$\begingroup$

Regarding the loss of Cas9 activity, you are already touching on the answer in your question. Cas9 as a nuclease/enzyme is always active, and will continue to cleave double-stranded DNA as long as there are complementary target sites and guide RNAs to guide it there. If the target site is not mutated or completely removed after cleavage, Cas9 can indeed cleave the same site again.

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.

I'm not sure if I misunderstand the point you are trying to make here, but the introduction of mutations is usually enough to prevent recurrent cleavage of the same site (although studies of off-target effects clearly show that this is not always the case):

Both Streptococcus pyogenes and Staphylococcus aureus Cas9 cleave their target sites between positions -3 and -4, counting from the end of the 20-nt guide sequence (see attached figure below). The double-stranded break can then be repaired either via non-homologous end joining (NHEJ), or via template-driven homologous recombination (HR).

CRISPR/Cas9 cleavage

In the former case (NHEJ), double-stranded breaks are sometimes repaired with small errors. These errors usually accumulate near the break site (read about NHEJ to understand why), and such mutations will typically destroy the gRNA recognition site. Note, however, that not all (in fact, very few) NHEJ repair events will result in the introduction of mutations, and "perfectly repaired" breaks both can and will be re-cleaved by Cas9. But since repeated cleavage is lethal to the cell, there is a selective advantage for mutations that prevent re-cleavage of a target site. Also note that the introduction of mutations (and thereby gene editing) via NHEJ is a random process, with no direct control by the researcher.

In the latter case (HR), the DNA template used for repairing the double-stranded break can typically be chosen by the researcher. It can for example be a chemically synthesized DNA fragment or PCR product with at least some sequence homology to regions flanking the cleavage site. But since there is no specific sequence requirement for the region in between the homology regions, this interspacing region can be designed such that it partly or completely replaces the original recognition and/or PAM sequence (violet region in figure below).

Repair of double-stranded break via HR

$\endgroup$
10
  • $\begingroup$ Thank you for your answer. I have confused two independent issues; "CAS's own activity" and "Once altered, can other target-genes be altered twice"? I understood the latter one, thanks to your answer. But, I think the first one is still not clear; for example, how many times does it take for a cell to divide to make the CAS docile? How can we measure the 'activity of CAS itself' at a given point in time? $\endgroup$ Nov 28 '20 at 20:06
  • $\begingroup$ When you write CAS (in capital letters), are you referring to Cas9? $\endgroup$
    – gaspanic
    Nov 28 '20 at 20:14
  • $\begingroup$ It's not clear to me what you're after with your first question. In what setting do you expect Cas9 to lose activity? Are Cas9 and gRNAs expressed or introduced into the cell via transfection/transformation? Like I tried to point out above, Cas9 itself doesn't lose its activity, and loss of DNA cleavage activity therefore boils down to knowing how long Cas9 and/or gRNAs exist inside the cell, i.e., their "lifetime" or half-life. Bacterial proteins are often very stable in eukaryotic cells and are not actively degraded, but I'm not sure if this is also the case for Cas9. $\endgroup$
    – gaspanic
    Nov 28 '20 at 20:44
  • $\begingroup$ Sorry the CAS means the Cas9. $\endgroup$ Nov 28 '20 at 21:05
  • $\begingroup$ To be exact, certainly that depends on how we get Cas9 into the cell . If the Cas9+gRNA complex itself is introduced into the cell, the problem is how long does it takes for all Cas9 to lose its activity from the point of introduction them into the cell. Perhaps it would be more reasonable to use a half-life, as it is difficult to say "the time until everything loses its activity". If you are using a vector expressing Cas9, then all Cas9 will not lose their activity until the last born Cas9 loses its activity. $\endgroup$ Nov 28 '20 at 21:18

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.