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!

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the comments below.