The CRISPR-Cas9 system
is a tool for cutting DNA
at a specifically targeted location.
The technique has already revolutionized gene editing
but scientists are always looking
for new possibilities,
so what else can CRISPR do?
Since being discovered in a bacterial immune system
CRISPR-Cas9 has been adapted
into a powerful tool for genomic research.
There are two components to the system:
a DNA-cutting protein called Cas9
and an RNA molecule known as the guide RNA.
Bound together, they form a complex
that can identify and cut specific sections of DNA.
First, Cas9 has to locate and bind
to a common sequence in the genome called a PAM.
Once the PAM is bound,
the guide RNA unwinds part of the double helix.
The RNA strand is designed
to match and bind a particular sequence in the DNA.
Once it’s found the correct sequence,
Cas9 can cut the DNA –
its two nuclease domains each make a nick
leading to a double strand break.
Although the cell will try to repair this break,
the fixing process is error-prone
and often inadvertently introduces mutations
that disable the gene.
This makes CRISPR a great tool
for knocking out specific genes.
But making double strand breaks
isn’t all CRISPR can do.
Some researchers are deactivating
one or both of Cas9’s cutting domains
and fusing new enzymes onto the protein.
Cas9 can then be used to transport those enzymes
to a specific DNA sequence.
In one example, Cas9 is fused to an enzyme,
a deaminase, which mutates specific DNA bases
– eventually replacing cytidine with thymidine.
This kind of precise gene editing
means you could turn a disease-causing mutation
into a healthy version of the gene
or introduce a stop codon at a specific place.
But it’s not all about gene editing.
Several labs have been working on ways to use CRISPR
to promote gene transcription.
They do this by deactivating Cas9 completely
so it can no longer cut DNA.
Instead, transcriptional activators are added to the Cas9
by either fusing them directly or via a string of peptides.
Alternatively, the activators can be recruited
to the guide RNA instead.
These activators
recruit the cell’s transcription machinery,
bringing RNA polymerase and other factors
to the target and increasing transcription of that gene.
The same principle applies to gene silencing.
A KRAB domain fused to the Cas9
inactivates transcription by recruiting more factors
that physically block the gene.
A more outside-the-box idea for using CRISPR
is to attach fluorescent proteins to the complex
so you can see where particular DNA sequences
are found in the cell.
This could be useful for things like visualizing
the 3D architecture of the genome,
or to paint an entire chromosome
and follow its position in the nucleus.
CRISPR has already changed the face of research
but these new ideas show
that what’s been achieved so far
could just be the tip of the iceberg
when it comes to CRISPR’s potential.
Whatever comes next,
it seems the CRISPR revolution is far from over.