I need help
here's the problem
I am trying to figure out how I should eliminate a specific gene in a eukaryotic cell
for a research project to see what the phenotype will be if the DNA lacked the protein
I have two options, I can use RNA Interference or the CRISPR
And to help me, I am going to make a venn diagram
so lets get to it
here's my Venn diagram, so lets get started
One difference is that RNAi targets RNA and CRISPR targets DNA.
RNA interference uses a dsRNA produced in a lab and it is cleaved by dicer, an enzyme
that turns the dsRNA into either siRNA or miRNA, which is around 21 nucleotides long.
From there, the modified RNA is binds to a silencer, called RNA induced silencing complex or RISC.
This inhibits the translation of the miRNA or siRNA
CRISPR uses a guide mRNA, created in a lab.
The guide mRNA binds to the Cas9
enzyme to recognize and cut the desired target sequence or gene of the DNA.
DNA then repairs the break naturally by copying template DNA and insetting it in the break.
The template DNA can be supplied either by the cell or researchers
but since we are only looking of the effects of inhibiting one gene, we would let the cell fix the break
I just mentioned a few more differences
so lets add them
RNAi uses dsRNA
Here are the differences I just said why explaining the two methods
Another difference is that RNAi's affects are only temporary
since it is only being silenced, the genes are still present and the phenotype is only being masked
since CRISPR deleted the sequence, the results are permanent
RNAi is also a much more simpler and quicker process than CRISPR
So I would have my results sooner, but time isn’t an issue for me.
Another difference is the amount of errors that can occur during gene editing.
The complete removal of the gene or genes during CRISPR results in less off target effects.
Some may still occur, but it is more likely to occur in RNAi
In RNAi, the silence of the gene can silence other proteins and genes that affect the phenotype.
I only want to see what happens when my desired gene is removed, and no other changes occur.
CRISPR is also newer than RNA interference. CRISPR was invented in the 2000s while RNA interference was invented in the 1980s
However both methods used a modified RNA to inhibit gene expression
Another similarity is that both methods can also result in false positives and off target effects
CRISPR can have off target effects if a mutation occurs when the cut from Cas9 is being fixed by the cell.
The cell can either glue the ends of the two broken pieces together, or insert a template.
If the cell decides to stick the ends together, mismtacthcing may occur and result in a mutation.
So, I decided that I am going to be using CRISPR method
and that's just because that it is more percised
even though it takes longer
But I don't want any off target affects or additional mutations
I just want to see what happens to the phenotype when I remove that one gene
So thank you guys so much for the help!
