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Non Life Sciences => Chemistry => Topic started by: scientizscht on 27/04/2019 13:52:49

Title: How does CRISPR/cas9 work?
Post by: scientizscht on 27/04/2019 13:52:49
Hi

I am having extreme difficulty to fully understand how crispr/cas9 works.

I have seen a ton of diagrams online but I think they don't fully explain how this works.

Can anyone explain please?

So far, I have identified the major components of the process: gRNA, DNA, cas9 enzyme.

How do these interact?

Thanks
Title: Re: How does CRISPR/cas9 work?
Post by: scientizscht on 17/05/2019 16:49:14
Anyone please?
Title: Re: How does CRISPR/cas9 work?
Post by: chiralSPO on 17/05/2019 19:57:54
no
Title: Re: How does CRISPR/cas9 work?
Post by: scientizscht on 17/05/2019 22:29:27

You mean 'not you'
Title: Re: How does CRISPR/cas9 work?
Post by: evan_au on 17/05/2019 23:54:26
Quote from: scientizt
You mean 'not you'
I think the intent was 'not to you'.

There are many people here who could explain it to others...

1. First I'll try with a simple analogy:
The "Search and Replace" function in a word processor has a template of the thing it is looking for. It scans through the document looking for a match to that template. When it finds a match, it replaces the matched string with the replacement string, and then continues searching.

CRISPR/CAS9 is like a Search and Replace function for DNA.

2. Now I'll refine the analogy:
CRISPR/CAS9 is like a Search and Replace function for DNA, except:
- It's not very accurate with the matching; it will sometimes declare a "match" when 10% of the bases don't match its search string. So it sometimes changes areas that weren't intended.
- It doesn't proceed methodically through the entire DNA string - it's like 100 editors starting at random parts of the DNA, and processing whatever they find near there. And some parts of the DNA are tightly bound up and not so accessible.
- When it makes a substitution, it is not entirely exact, so it sometimes changes it to something different from what is intended.
- Even when it makes a change, the cell's virus detection system is at work. It's as if "the cloud" is trying to repair your document while the search and replace is in progress.
- Sometimes the cell's virus defence system realises that it has lost the battle, and commits suicide (apotosis). It's like "the cloud" realises that your document is corrupted, and puts it in the recycle bin.
- It has been seen that those cells where CRISPR/CAS9 succeed tend to have a defective detection system for viruses (and mutations in general).


3. Now I'll try with some details:
https://en.wikipedia.org/wiki/CRISPR_gene_editing
https://en.wikipedia.org/wiki/CRISPR
https://en.wikipedia.org/wiki/Cas9
https://en.wikipedia.org/wiki/Genetic_engineering

If you can't understand these three (quite different) levels of detail, then at least you will understand why it can't be explained to you...
Title: Re: How does CRISPR/cas9 work?
Post by: scientizscht on 18/05/2019 00:29:31
Interesting but how does it work exactly?

You have a gRNA sequence and you bind the Cas9 to it.

That gRNA will go and bind to specific DNA sequence.

Then the Cas9 will cut the DNA (at which point?).

And how it will actually insert a given new sequence? Will it use part of the gRNA?
Title: Re: How does CRISPR/cas9 work?
Post by: RubyO on 21/05/2019 10:35:29
Quote
Then the Cas9 will cut the DNA (at which point?)

The gRNA is typically 20 letters long, and Cas9 cuts 3 letters from the end, or 17 letters in.

Quote
And how it will actually insert a given new sequence? Will it use part of the gRNA?

Cas9 itself doesn't insert a new sequence, it just cuts the DNA. DNA with breaks in it is very dangerous for cells, so the cell's own repair mechanisms join the DNA back together. It has two ways of doing this:

1. Just stick the two ends back together - this often involves adding or removing a few letters at random from the break point, so it can lead to mutations. It's not ideal for the cell, but it's better than nothing if option 2 isn't available. If you, as a scientist, just want to mutate a gene so it stops working, this is a good way to do it.

2. The better way for the cell is to use an unbroken DNA copy of the same sequence, to make sure the sequence isn't changed in the repair. Cells don't always naturally have a copy to use, depending on where they are in their replication cycle, which is why option 1 also exists. But if you, the scientist, want to cause a specific change, when you give the cell the Cas9 and gRNA, you also give it a piece of DNA with the sequence you want to end up with. The cell can use this DNA as a repair template and integrate your new sequence during the repair.
Title: Re: How does CRISPR/cas9 work?
Post by: scientizscht on 21/05/2019 12:31:19
That's very insightful thanks.

1) when does the CRISPR-gRNA bind to the DNA? Does the cell need to be in mitosis or it binds anytime?

2) how does the whole thing enter the cell?

3) what is the rate of success of editing a specific gene among eg 100 cells in vivo?