Are you looking to perform a gene silencing
project? Should you use CRISPR, RNAi, or TALENs to
get the job done? In this video, we'll explain how each system
works and how they differ in terms of experimental set-up, efficiency, off-target effects, and
more. So you want to do a gene silencing project! The first question to ask yourself is: are
you looking to knockout or knockdown a gene? A gene
knockout is when a gene has acquired a frameshift mutation such that the cell no longer expresses
any functional protein. When a double stranded break in the DNA is
created, the cell can repair it via the Non-Homologous End Joining or NHEJ repair pathway. This process creates insertions or deletions
that can cause a frameshift mutation that eliminates gene expression. CRISPR and TALENs can be used for gene knockouts. A gene knockdown is when gene expression is
reduced but not completely eliminated. This is typically done by degrading or blocking
translation of the gene's mRNA transcript. Some mRNA may still escape regulation so there
is still low level gene expression. RNAi can be used for gene knockdowns. Let's dive deeper into the different systems,
starting with CRISPR! For more details about CRISPR, visit our knowledge
base that we've linked in the top right corner. In a nutshell, the basic CRISPR system has
two components: a single guide RNA (sgRNA) and a Cas9 nuclease. The sgRNA forms a ribonucleoprotein with Cas9,
guiding it to a specific target sequence, using the 20bp at its 5' end. Cas9 must also recognize a short sequence
adjacent to the target sequence called the PAM sequence which differs depending on the
species of Cas9. Once docked, Cas9 will create a double stranded
break in the DNA. The TALEN system consists of a transcription
activator-like (TAL) protein that is fused to the FokI nuclease. Each of the 33 repeats in the TAL DNA-binding
domain can differ by two amino acids, which determines which nucleotide it will bind. By combining 12-31 of these repeats, a TALEN
can be engineered to bind to a specific DNA sequence. Two TALENs must dimerize in order to create
a double stranded break in the DNA. For CRISPR and TALENs, the cell can repair
the double stranded DNA via NHEJ repair pathway. Then selection must be carried out to isolate
the cells with the frameshift mutation leading to gene knockout. In the RNAi gene knockdown method, short RNAs
such as siRNA are designed complementary to the target mRNA. One siRNA is loaded into the RNA-induced silencing
complex or RISC which guides the system to bind and cleave the target mRNA, resulting
in gene knockdown. If binding is imperfect, mRNA translation
will only be inhibited. So now that you know the basics of how CRISPR,
TALENs, and RNAi work, let's compare ease of experimental design, efficiency, off-target
effects, flexibility and applications. Ease of design. In terms of ease of design, siRNAs are easiest,
followed by sgRNAs for CRISPR, with TALENs being the most labor intensive. siRNAs can be designed to target almost any
mRNA at any locus. The CRISPR targeting system is more restricted
as sgRNAs must be designed for DNA sequences that are adjacent to PAM sequences. Finally, TALENs are also used in pairs so
there would be double the design work. Ease of experimental set-up. In terms of ease of experimental set-up, siRNA
are also easiest, followed by CRISPR, followed by TALENs. siRNA need only be delivered as one transgene
and utilizes the cell's host machinery to achieve detectable gene knockdown in only
24 hours. CRISPR requires the delivery of not only sgRNA
but also the Cas9 nuclease. Because of this, it may be difficult to use
smaller viral expression systems such as Adeno-associated Viruses. TALENs are
even more difficult to clone as they have larger, repetitive sequences and require double
the cloning work as they must be used in pairs. Ease of experimental validation. In terms of experimental validation, different
techniques are available depending on the system you use. With RNAi, you can assess gene knockdown by
measuring mRNA levels using qRT-PCR and protein levels using Western Blot. If mRNA levels are decreased but protein levels
remain the same, protein turnover may simply be slow. If mRNA levels remain the same but protein
levels decrease, the siRNA may be inhibiting translation rather than degrading mRNA. Experimental set-up typically requires 1-2
days, however, an antibody for western blot may not be available. With CRISPR and TALEN systems, the % of edited
cells can be estimated via the Mismatch Cleavage Detection Assay, or more commonly known as
the Surveyor Assay. In this method, target DNA from the cells
is amplified via PCR then denatured and reannealed, forming hybrids between unedited and edited
strands in the process. Any hybrids with mismatches are then cleaved
using the Surveyor or T7E1 nuclease. Results are run on a gel to estimate the % of
edited cells. A monoclone must then be isolated and validated,
using Sanger Sequencing to verify the frameshift mutation, all of which add days to the project. The efficiency of each system depends on many
factors so it's difficult to directly compare RNAi, CRISPR, and TALENs. In general, efficiency is less important for gene knockout
than for gene knockdown. A low efficiency knockout for CRISPR and TALENs
simply means that more clones will need to be screened to find a monoclonal cell line
with the gene completely silenced. A low efficiency knockdown for RNAi, however,
indicates less gene repression and less pronounced phenotypes. TALENs have the lowest off-target editing
effects followed by CRISPR and then RNAi. With TALENs, there is a low chance of another
site possessing the two opposing target sites required for the two TALENs to bind independently. CRISPR off-target effects can be reduced when
Cas9 nickase is used. Cas9 nickases are modified such that the Cas9
can only cleave one DNA strand. So two sgRNAs targeting opposite DNA strands
are required to make a double stranded cut. RNAi, on the other hand, can cause significant
off-target effects. One siRNA can potentially repress hundreds
of off-target mRNA transcripts as it doesn't require strict sequence complimentarity to
bind. CRISPR is the most versatile system for gene
manipulation, followed by TALENs and then RNAi. CRISPR can easily be adapted for gene knockdown,
knockout, knock-in, activation, repression, base editing, or imaging. To achieve a gene knock-in, simply provide a repair template. The cell will repair the DNA break via the
homology directed repair pathway using the repair template to incorporate the new sequence. By modifying the Cas9 into an enzymatically
"dead" Cas9 that can't cut DNA, and fusing it to various effector proteins, the CRISPR
system can also be used to target and activate, repress, or image genes. Similarly, TALENs can also be fused to effector
proteins to further expand its versatility. On the other hand, RNAi can only be used for
gene knockdown. Finally, in terms of applications, which gene
silencing method is best to use depends on the project goal. If the goal is to study a gene's function,
knocking out a gene using CRISPR or TALENs results in a more dramatic change in phenotype
vs. knocking down a gene using RNAi. If, however, knocking out the gene results
in cell death or reduced cell fitness, it may be more suitable to use RNAi. If the goal is to study a mutation associated
with a genetic disease, CRISPR or TALENs are able to introduce genetic mutations whereas
RNAi cannot. If the goal is to do a high-throughput screening
project, CRISPR or RNAi systems scale easily for each target sequence. On the other hand, creation of TALEN libraries
are much more labour and cost intensive to design and clone due to the large size of
TALENs and their repetitive elements. There are many more specialized applications
where one system is more suitable over another, and you can learn more about this by visiting
our knowledge base linked below in the description! We hope this video helps you determine which
system is best for your gene silencing project! Please don't forget to subscribe to our channel and
please feel free to leave your questions in the comments below.
