Let’s say you’re curious about the function
of a particular gene of interest in a specific
eukaryotic organism. A good way to determine
what a gene does is by looking at situations
in which the normal function of that gene
is altered. One way you could do this is by
looking at organisms with naturally occuring
mutations in that gene. But say your gene
of interest doesn’t have any of these mutations,
how then do you observe alterations in your
gene’s expression? Well, there are two ways
to silence your gene by limiting or completely
eliminating its expression. Two powerful ways
to do this are through RNA interference and
CRISPR.
RNA interference occurs naturally in eukaryotic
cells and serves as a mechanism to limit the
invasion of foreign genes and control the
expression of the cell’s own genes. We are
able to take advantage of this naturally occurring
process by injecting the double stranded RNA
of our gene of interest into the cell. What
happens is the cell recognizes the injected
double-stranded RNA as a foreighn body and
seeks to destroy it as it evolutionarily would
have done to protect itself from foerign invaders
like viruses. So how does it do this? The
invading double-stranded RNA is cut by the
enzyme Dicer, an endonuclease protein. This
forms siRNAs that then bind with proteins,
including Argonaute, to form an RNA induced
silencing complex, or RISC. After one strand
from the double-stranded RNA is chosen as
a guide RNA, the RISC complex looks for its
complementary mRNA. It then represses or degrades
the translation of the mRNA.
In response to viruses, prokaryotes produce
two types of short RNA that form a complex
with the CAS 9 protein. One’s sequence matches
the virus sequence. CAS 9 is
a nuclease that can cut DNA. Once
again similar to the eukaryotic analog, the
guide RNA finds the target in the virus’s
genome and CAS 9 cuts the target DNA. When
this process is modified by being introduced
to eukaryotic DNA, the cell will try to repair
the cut DNA. While this process is imperfect
and typically results in mutations that can
be advantageous to studying the gene’s function,
an even more useful result can be produced.
If a piece of DNA carrying the desired sequence
is added, it can be incorporated into the
genome, allowing scientists to “cut and
paste” genes.
So which is the better method, RNAi or CRISPR?
While that may depend on what you are specifically
looking at, in general I would say that there
are more advantages to using CRISPR which
is probably why it is a more commonly used
tool in research today than RNAi. One major,
and probably obvious, advantage to CRISPR
is that it edits DNA, making perminant changes
in living cells, while RNA interference only
makes temporary changes. Furthermore, CRISPR
allows you to insert genes and target many
genes at once. For those reasons I believe
CRISPR is a more powerful tool and I would
choose it to investigate my gene of interest.
