Let's say you're studying the function of a particular protein.
You have some cells growing in culture
and you want to tinker with your protein in those cells to tease apart what it does.
You might want to make sure your cells can't produce that protein anymore.
Or you could introduce a particular mutation in your protein
or attach a fluorescent tag to be able to see
where your protein goes inside the cell.
You can do this by introducing siRNA into the cell to knock down the protein
Or transfect the cells with plasmid DNA to express your protein with a mutation or a GFP tag
These methods work just fine and scientists have been using them successfully for decades.
But they have some downsides.
siRNA has some issues with off-target effects
and when you transfect in a plasmid it's very difficult to control exactly how much protein you introduce.
Plus, both methods only modify your cells for a few days.
A much more elegant approach would be to edit the gene for your protein directly in the cell's own DNA.
That way you know for certain that the cells will produce your mutated protein at its natural levels
which makes for a much cleaner comparison in your experiment.
Over the last few years this has become possible using a technique called CRISPR-CAS9
It uses the DNA-cutting enzyme CAS9 from the bacteria Streptococcus pyogenes
bound to a guide RNA
which can be very specifically targeted towards a bit of DNA
allowing CAS9 to cut it in a very specific place.
In order to cut a section of DNA
CAS9 needs to find it first, and for that it needs two things:
Firstly, the target DNA needs to have a so-called PAM site
which is a short sequence of any nucleotide followed by two Gs.
But these PAM sites are extremely common in the genome
so CAS9 needs something a bit more specific to find the right place to cut the DNA.
To do this it uses a 20 nucleotide section of the guide-RNA
that is complementary with the bit of target DNA just before the PAM sequence.
When everything is bound together in the right position
the two nuclease domains in CAS9 cut the DNA in both the non-complementary and complementary strand.
You're then left with a double strand break in the DNA exactly where you want it to be
and depending on what kind of result you're after, you can then do three things to fix that DNA.
The first thing is by doing nothing
and letting the cell fix the double strand break itself.
But cells are rubbish at fixing double strand breaks
so will often end up accidentally inserting or deleting bits of DNA.
These indels mean that the order of the sequence can get pushed out of the reading frame
and the cell will stop making your protein.
If you want to keep your protein but modify it with a mutation or a tag
you'll need to provide the cell with template DNA of how to fix the double strand break.
This template can either be a tiny single strand DNA oligonucleotide with a point mutation in it
or an extended piece of double stranded DNA template to create an insertion for something like a GFP tag.
So CRISPR-CAS9 allows us to do all kinds of sophisticated editing in the cell's own genome
and with so many different applications, this elegant technique is already changing the way we do biomedical research.
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