This video will discuss two gene editing mthods:
RNAi and CRISPR
RNAi
Is a method used for gene knockdown which
is when gene expression is reduced but not
completely eliminated.
This process is done through blocking translation
of the gene mrna transcript
RNAi utilizes short RNA’s such as siRNA
or miRNA which are designed complementary
to the target Mrna sequence
Then the siRNA is loaded into RISC or RNA-Induced
silencing complex which guides the system
to bind to and cleave or inhibit the target
Mrna resulting in the knockdown of that gene
or limited expression of it
CRISPR
CRISPR is a gene editing method used for gene
knockout which is when a gene experiences
a mutation that causes the cell to no longer
express any functional protein
CRISPR utilizes two components for gene knockout.
A single guide RNA (sgRNA) and a case9 nuclease.
These two components join to form a ribonucleprotein
that target a specific sequence adjacent to
a sequence known as PAM which is a short dna
sequence used to identify the targeted area
Once it binds to the target sequence, cas9
cutts the desired sequence creating a double
strand break in the DNA which is later ligated
together.
When the break is repaired, new mutations
occur to the sequence causing a disruption
to the reading resulting in gene knockout.
Decision
Lets pretend we have a cell that contains
gene A and we want to study the impacts of
not producing gene A, which method is best
to use?
The use of the RNAi method is speedier as
siRNAs can be delivered as one transgene to
achieve gene knockdown
and more flexible as they can target any Mrna
sequence at any locus,
while CRISPR can take longer as it needs to
deliver both sgRNA and cas9 nuclease to the
mrna CRISPR is more restricted as sgRNA must
be designed for DNA sequences adjacent to
PAM
Even though CRISPR is more time consuming
yet its more efficient, results in less off-target
effects, and has better chances of eliminating
the gene than RNAi.
Furthermore CRISPER has a wide range of application
meaning it can be used in several ways such
as fix an allele or replace it.
In order to understand how gene A impacts
the cell, completely eliminating the gene
is more beneficial as it provides more dramatic
changes in phenotype giving more insight on
the function of gene A
And that will conclude the video thank you
for watching
