when studying the effect of not
producing or producing very little of a
protein from gene A in my favorite
eukaryote I can take one of two possible
approaches: the CRISPR method or the RNA
interference method. CRISPR stands for
clustered regularly interspaced short
palindromic repeats and it's a type of
genetic editing that allows me to modify
DNA genetic code so that I can prevent
transcription of a target gene sequence.
This method uses a guide RNA sequence
complementary to the DNA of gene A as
well as a cas protein, usually Cas9
Together they make up the CRISPR
Cas9 complex. The guide RNA acts as
a GPS to bring the CRISPR-Cas9
complex to the DNA of gene A in the
nucleus. Once there, the Cas9 protein
cuts the DNA at gene A rendering it
untranscribable and turning off gene
A. When this happens, the cell will try
to repair the DNA, but mutations often
occur which keeps the gene off. For
additional control, researchers often
inject DNA templates with sequences
complementary to the cut pieces of the
DNA. In such a case the cell will repair
itself using the researcher's DNA
template rather than making its own. Next
we will talk about the RNA interference
method.  This is a type of genetic editing
that interferes with mRNA, preventing
translation of a target gene sequence.
This system is made of two RNA fragments:
small interfering RNAs and micro RNAs
both of which are created when a protein
called DICER chops up larger
double-stranded RNA strands. The RNA
fragments associate with proteins to
form a complex called RISC and search
for their complimentary strands in the
cell. In my case it looks for the mRNA
strand for gene A. When the target mRNA
is found, the mRNA is cut preventing its
translation and silencing the specified
gene. Sometimes the micro-RNA sequences
only partially match the mRNA sequence.
In such a case the mRNA strand will not
be cut but RISC remains on the mRNA
physically blocking the translation.
Quick little recap, RNA interference
targets RNA while CRISPR targets DNA. RNA
interference is also a temporary masking
of the phenotype and the gene can
sometimes produce a small amount of the
protein, while CRISPR is more permanent
and complete. However RNA interference is
more likely to have off target results
because
micro-RNA can sometimes partially bind
to other mRNA sequences. CRISPR allows
for greater control since it eliminates
the DNA sequence for only that gene. Both
methods use guide RNA to get to the
desired target and they both use enzyme
complexes to cut their target. Having
said all that, I would use RNA
interference. We are talking about my
favorite eukaryote here, so I really like
the fact that RNA interference allows me
to reverse my actions and put my
favorite eukaryote back to normal. CRISPR
is also a lot more expensive and
complicated than RNA interference and
since I don't like spending a lot of
money and I don't like things that are
complicated RNA interference appeals to
me a lot more.
