Science Buddies: "Ask an Expert"

Hi,
I'm taking part in a science competition, and I'll most likely be explaining how CRISPR can revolutionize people's lives in the future. To do this I must have a thorough understanding of the topic. I have conducted some research, but I've developed many questions regarding how exactly the system functions:
Does the Cas9 enzyme cut double or single strands? Or can it do both? Does it simply cut at one location or does it cut out a chunk of DNA?
Correct me if I'm wrong: CRISPR uses the guide RNA to locate a DNA segment, cuts it, then two repair pathways are present: Non-homologous end joining or homology directed repair, most commonly, homologous recombination. The Cas9-RNA complex also brings a bit of DNA repair template which triggers the homologous recombination (are meiotic and mitotic recombination divisions of homologous recombination? If so, how do they differ, and which type of repair mechanism is present during gene editing with CRISPR?) and uses the repair template to change/add genes (This goes back to the question of how the enzyme cuts the DNA - does the cell recognize bases on both ends of the repair template and gets fooled into using it to replace the missing piece of genetic code that has been cut out, if it has cut out a chunk? Or something else?).
I remember reading about homologous recombination occurring during meiosis, but can it also occur in non-dividing cells if there is a repair template present, and is that what enables scientists to edit the genome, to trigger this repair mechanism and replace broken bits with a modified piece with recognized bases on both ends, brought into the cell by an enzyme like Cas9?
In addition, I looked into knockdown/activation involving CRISPR-Cas9 on wikipedia, and I understand how they fundamentally use a dead Cas9 to not cut genes but let them sit there and shuts down the gene, but then I came across this sentence: The targeted site is methylated, epigenetically modifying the gene. This modification inhibits transcription.Is there any relatively simple way to explain this? Of course, I wouldn't be required to fully explain how because of [details on knockdown/activation], this allows scientists to investigate the effects of certain genes...blah blah blah, but I do think understanding it could be very beneficial.
I'd really appreciate it if someone could explain this in a way that's understandable, meaning all jargons are explained without introducing more jargons... Thanks!!!

Hi, there! CRISPR is a GREAT topic for a science fair project, and you have some excellent questions. Hopefully I can help clarify a few things for you.

Does the Cas9 enzyme cut double or single strands? Or can it do both? Does it simply cut at one location or does it cut out a chunk of DNA?
- There are different varieties Cas9 enzymes. Some make single strand cuts, or "nicks," and some make double-stranded breaks. But a single Cas9 enzyme can't make both kinds of cuts.
- Either way, a Cas9 molecule can cut at any location in the genome that it is brought to by the guide RNA. The more places in the genome match up with the guide RNA, the more places Cas9 can potentially cut.

Correct me if I'm wrong: CRISPR uses the guide RNA to locate a DNA segment, cuts it, then two repair pathways are present: Non-homologous end joining or homology directed repair, most commonly, homologous recombination. The Cas9-RNA complex also brings a bit of DNA repair template which triggers the homologous recombination (are meiotic and mitotic recombination divisions of homologous recombination? If so, how do they differ, and which type of repair mechanism is present during gene editing with CRISPR?) and uses the repair template to change/add genes (This goes back to the question of how the enzyme cuts the DNA - does the cell recognize bases on both ends of the repair template and gets fooled into using it to replace the missing piece of genetic code that has been cut out, if it has cut out a chunk? Or something else?).
- You are exactly right that the guide RNA brings the Cas9 to a DNA sequence to cut it, triggering either non-homologous end joining (NHEJ) or homology-directed repair (HDR).
- However, the Cas9-gRNA complex is not associated with a DNA repair template. Scientists are currently working on this type of repair by introducing a repair template along with Cas9 and the gRNA, but the efficiency of HDR is very low, while the efficiency of CRISPR is quite high. This means that in cells expressing Cas9, a gRNA, and a repair template, there will be a mix of cells repaired by both HDR and NHEJ. More research is needed to optimize the use of CRISPR for gene editing by repair template insertion.
- I believe homologous recombination does underlie both mitotic and meiotic recombination.

I remember reading about homologous recombination occurring during meiosis, but can it also occur in non-dividing cells if there is a repair template present, and is that what enables scientists to edit the genome, to trigger this repair mechanism and replace broken bits with a modified piece with recognized bases on both ends, brought into the cell by an enzyme like Cas9?
- Homologous recombination requires a sister chromatid (or other homolgous template), so it normally only occurs in dividing cells. However, you are right that it should be able to occur in non-dividing cells with a repair template.

In addition, I looked into knockdown/activation involving CRISPR-Cas9 on wikipedia, and I understand how they fundamentally use a dead Cas9 to not cut genes but let them sit there and shuts down the gene, but then I came across this sentence: The targeted site is methylated, epigenetically modifying the gene. This modification inhibits transcription.Is there any relatively simple way to explain this?
- In this case, the Cas9 is indeed "dead" in that it cannot cut the genomic sequence that the gRNA brings it to. However, the Cas9 here is fused to another enzyme (such as a methyltransferase), and it is this enzyme that acts on the target DNA. So the idea is methyltransferase -> linked to dead Cas9 -> targeted in genome by gRNA -> putting methyltransferase close enough to targeted DNA sequence to epigenetically silence it.

I hope that helps, and feel free to ask if something still isn't clear!

Thanks a lot! I will take a closer look tomorrow and see if I have any further questions as it is getting late, but looks very promising!