Today we are going to discuss the
Elimination and Reduction of gene expression in the Platypus.
We are specifically concerned with what is called Gene A,
and we want to investigate how to eliminate
or reduce Gene A's expression.
Our first method is the CRISPR approach, which stands for Clustered Regularly
Interspaced Palindromic Repeats
and is a naturally occuring prokaryotic
process to defend against viruses. When
a viral DNA is detected, the cell will make short RNA
which contains a matching sequence to the invading DNA. This matching
RNA, known as guide RNA
binds with a protein called Cas9, forming the CRISPR complex to cut
and disable DNA.
We start with guide RNA binding to
the Cas9 protein, which forms the CRISPR complex.
When the guide RNA finds the matching sequence on the
Target DNA, it will
bind to a short sequence, called the PAM sequence, followed by Cas9
unzipping the target DNA, so that the guide RNA can bind with the target.
Once the match is complete, the target DNA is cleaved, and
the cell attempts to
repair the DNA, but the process is prone to errors, and the gene
is disabled. Our second method is RNA Interference,
also known as RNAi, and is a Eukaryotic process
that uses small double stranded RNA, taking
place in the Cytoplasm.
There are two main types of small RNA.
Small Interfering RNA, known as siRNA, which comes
from either mRNA, transposons, or some viral introduction
and targets genes that it comes from.
We also have MicroRNA, known as miRNA, which is
going to be transcribed from DNA in the nucleus, targeting other genes.
With miRNA, we begin with
DNA in the nucleus transcribed into Pri-miRNA, which
is the cleaved, leaving hairpins with short RNA.
We then see a protein called Dicer, which removes
the loop of the hairpin, leaving small strands of RNA.
Next, and Argonaute Protein combines with one
of the miRNA strands called a RISC,
or RNA Induced Silencing Complex. Which then binds with specific
mRNA. miRNA tends to
bind imperfectly to the target site,
which can result in result in either the cleavage of mRNA to be degraded
or translation simple being inhibited.
miRNA's imperfect binding
makes it able to bind to hundreds of different mRNA.
With siRNA,
we see a similar mechanism, beginning with double
stranded RNA. Which is then cut by Dicer,
followed by binding to an Argonaute Protein
to form a RISC complex. This time,
we see perfect binding to the target site on the mRNA.
Meaning that the mRNA will be cleaved and degraded
rather than having translation inhibited like in the case of miRNA.
To compare, CRISPR uses DNA-level
action to silence genes, with RNAi using RNA-level
action. We can also see that CRISPR is a fairly permanent process
with gene silencing, whereas RNAi
can simply reduce expression. With CRISPR
being the more precise of the two.
Our method of choice is RNAi,
allowing for the reduction of gene expression to desirable
levels to study different extents of expression.
The complete gene silencing associated with CRISPR
may kill the cell if the gene is crucial for survival.
As a result of our reduction in Gene A, we have seen the resulting inability
of the male platypus to produce its natural venom.
This may make competition with other males for a mate increasingly difficult.
