We are at the dawn of a new and empowered age of genetic engineering.
And ushering our entry into this exciting era is the revolutionary tool called CRISPR
or clustered regularly interspaced short palindromic repeats, originally discovered as part of
the bacterial immune system.
But the true brilliance of the CRISPR system is that its applications extend so much further
beyond simply gene editing itself.
As you will learn in this video, scientists have also been cleverly modifying the CRISPR
system to reversibly activate or silence genes as well as edit the epigenome in ways that
were previously unimaginable.
First, let's quickly review how CRISPR gene editing works.
The mechanism is elegantly simple, requiring only two components: a single guide RNA targeted
to your gene of interest and the RNA-guided Cas9 endonuclease.
In the presence of an sgRNA, Cas9 endonuclease homes in on the target sequence and makes
a double stranded cut directly upstream of a protospacer-adjacent-motif, or PAM sequence.
This sequence is NGG for the Cas9 protein of Streptococcus pyogenes.
After the double stranded cut is made, the cell can repair the cut using either the non-homologous
end joining repair pathway or the homology directed repair pathway.
In the NHEJ pathway, the ends of the DNA are ligated back together, often resulting in
random small insertions or deletions that can effectively disrupt gene expression.
In the HDR pathway, scientists can add a DNA repair template containing their desired sequence
for insertion into the target strand at the break site.
The list of CRISPR endonucleases that can be used for gene editing are numerous and
growing every year.
From smaller endonucleases that can fit into adeno-associated viruses for tissue-specific
delivery to endonucleases modified to reduce off-target effects, the CRISPR system has
unprecedented versatility.
But the edits made by CRISPR Cas9 are permanent which is not ideal for many applications and
can be toxic to cells.
This led to the development of a catalytically dead Cas9 called dCas9, a mutant in which
both cleavage domains of Cas9 are inactivated.
By itself, dCas9 can still target and bind DNA with high precision, however, instead
of irreversibly altering the genome, the binding of dCas9 reversibly interferes with and silences
gene expression.
And better yet, when dCas9 is fused to transcriptional regulators, it can be used to reversibly manipulate
gene expression levels!
What's more, this system works at the transcriptional level, and can activate or repress noncoding
RNAs, antisense transcripts and microRNAs.
These are all notoriously difficult to silence by classical CRISPR approaches and RNAi, but
they are no longer out of reach thanks to CRISPR.
Let's go through a few of the ways scientists have repurposed dCas9 for gene activation
and repression.
The dCas9-SAM system is a powerful tool for amplifying gene expression via the recruitment
of transcriptional activators to a targeted gene.
This is accomplished by fusing dCas9 to an array of transcriptional activators, such
as VP64 and p65, and using sgRNAs targeted to endogenous promoters.
This dCas9-SAM system can amplify gene expression by 10 to multiple thousand-fold.
This effect is so powerful that researchers are already using dCas9-SAM to eradicate HIV-1
in infected organisms.
When dCas9 is fused to the transcriptional repressor, KRAB, it can recruit histone modifiers
for 5-10 fold reversible repression of gene expression via the formation of heterochromatin.
As mentioned earlier, unlike the RNA interference method which relies on degradation of mRNA
in the cytoplasm for gene repression, dCas9-KRAB enables silencing at the DNA level, opening
up a new array of targets such as non-coding RNAs, microRNAs, antisense transcripts, and
nuclear localized RNAs.
Epigenetic gene regulation is an elegant series of chemical modifications that results in
the selective silencing or activation of particular genes without direct alteration of the DNA
primary sequence.
DNA methylation as well as histone tail modifications such as methylation or acetylation, regulate
whether a particular genomic sequence is stretched out and accessible to the transcriptional
machinery, or whether it gets tightly bundled into transcriptionally silent heterochromatin.
Thus, the epigenome acts as a second level of gene expression regulation, which is heritable,
but also reversible.
For years, researchers have been attempting to introduce specific epigenetic modifications
in an effort to adjust gene expression levels, which could have enormous therapeutic potential,
but their methods were laborious, unreliable and expensive.
dCas9 opens up a whole new world of possibilities for epigenetic studies.
Because it is so easy to attach and swap out different effectors to the dCas9 platform,
dCas9 can also be fused to histone or DNA methylation modifiers to make it an amazingly
versatile tool for epigenetic manipulations.
For example, dCas9 fused to the histone acetyltransferase, p300, is able to specifically
acetylate histones near the target DNA sequence, activating gene expression
from 50 to 10,000 fold.
Or, when dCas9 is fused to the demethylase, LSD1, it can demethylate histones near the
target sequence.
dCas9 fusion systems can also be used to introduce or erase DNA methylation.
DNA methylation controls everything from cell development to differentiation, and many diseases
are associated with improper DNA methylation patterns.
dCas9 fused to DNA-manipulating effectors enable scientists to study the effects of
DNA methylation-related silencing, and even develop therapies for combating disease.
The two best known dCas9 fusions that are currently available for manipulating DNA methylation
status are dCas9-Tet1 which demethylates DNA and dCas9-DNMT3A, which methylates CpG sites
in vivo.
These tools have already been used to investigate the effect of demethylation and hypermethylation
of tumor-suppressor genes, such as BRCA1 and CDKN1A.
In the near future, dCas9 fusions can be used to selectively turn genes on or off in an
effort to treat disease, making it a powerful and transformative tool to spur the development
of molecular therapeutics.
The dCas9 systems described in this video are only just the beginning of what will no
doubt soon be a full, and comprehensive suite of tools that could allow you to perform just
about any reversible DNA manipulation you can dream of!
To help you conduct your genomic and epigenomic research faster, smarter, and more affordably,
our abm team not only offers the Cas9 nuclease, nickase and an entire library of CRISPR knockout
cell lines and custom services, we will also offer an exciting new suite of dCas9 systems
you can add to your toolbox!
Just check in with our online store at www.abmgood.com,
click on "CRISPR gene editing tools",
and you'll be able to see all the amazing new tools we have to offer!
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For more information about the CRISPR dCas9 system, please visit our CRISPR Cas9 knowledge base.
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Thank you for watching!