dCas9: A New Identity for Cas9 in Cancer Epigenetics

How can we manipulate our genes, without changing the genome?

Humans have been pursuing a scientific solution to cancer, birth-defects, and biological radiation damage since the discovery of DNA in the 1950’s. This means that we’ve been undertaking genetics for less than 0.004% of mankind’s 200,000 year time on Earth.

In this minuscule amount of time we’ve discovered the instructions that dictates our very being (DNA), and we’ve even discovered how to edit these instructions as we desire (CRISPR/Cas9). Now we’re beginning to uncover how these instructions are getting coiled up, shifted around, and are even getting switched on/off.

As we push the limits of manipulation with the base pairs of the genome, we’re also pushing forward in an arguably more complex and unknown front: epigenetics.

If switching genes on and off to solve some of the future’s biggest problems sounds incredible to you, take a trip with me into the world of Epigenetics!

So what really is epigenetics?

We define epigenetics as the study of how DNA interacts with the millions of different biological molecules in the cell.

This is an extremely new field, having significant progress only being made after 2001. Through studying it, we are starting to learn the different actors at play, coming together to build us into unique, individual beings. Rather than working directly with the genome, epigenetics works with the epigenome.

The epigenome refers to the overlapping term for the series of chemical tags placed on DNA, as well as the physical coiling and placement of DNA in the nucleus of our cells.

These chemical tags alone don’t do much, but they act as signaling beacons for special proteins and enzymes which can affect the gene expression. These tags change the gene expression either by increasing the rate of gene expression (expression), or decreasing it (repression).

You can think of each class of chemical tag as a flower with a unique scent: different types of flowers will attract different bugs, depending on their unique smell.

Different bugs (proteins and enzymes) at different flowers (chemical tags), will oftentimes have very different interactions, and do very different things.

DNA Methylation

Methylation is one of the most prevalent of these chemical tags in epigenetics.

This chemical tag is the addition of a “Methyl” chemical group to the cytosine (“C”) base of DNA. When this chemical group is added to the cytosine, it usually acts as a signal for proteins which repress gene expression to come and do their thing. At the same time, the methyl group also interferes with proteins that increase gene expression, preventing them from interacting with the methylated area of the gene.

With these two properties, we usually associate DNA Methylation with the repression of gene expression. Consequently the removal of methylation (aka. demethylation), is usually associated with the subsequent increase of gene expression.

How is something Epigenetic?

Any molecule effecting gene expression may or may not be Epigenetic. To be considered epigenetic, the change must be:

  1. Non-Genomic: Any Epigenetic change must come about via molecules altering gene expression, and not actually changing the base pair arrangement or structure of the DNA Double Helix.
  2. Mitotically Heritable: When these changes to gene expression occur in the cell, the change must pass on when the cell divides. This accounts for why epigenetic changes can effect an organism for all their life, and even in their offspring!

Changing Cas9 to dCas9

Before getting into the details of how we can change up Cas9 and use it differently, I highly recommend my colleague Hannah’s article summarizing how CRISPR/Cas9 works.

Cas9 binding its DNA target

Cas9 on its own is an endonuclease enzyme, meaning that it will encapsulate a part of the DNA of interest, and then cut it off from the DNA chain.

This process starts when the Cas9 enzyme would be binded to a guide RNA. This guide RNA would bring the Cas9 enzyme to the target spot on the DNA, where it will cut the target DNA.

However if you get rid of Cas9’s distinct DNA-cutting ability, you’re left with an enzyme that can bind other RNAs and proteins, while still being able to encapsulate DNA. This enzyme is known as “Deactivated Cas9”, or dCas9.

dCas9 has the ability to bind to different enzymes and proteins which are responsible for increasing or decreasing gene expression, as well as a guide RNA just like regular Cas9. Using dCas9, scientists at Purdue University decided to see if they can increase the expression of a cancer suppressor gene, BRCA1.

The BRCA1/dCas9 Experiment

The Idea

In cancer tumors such as breast cancer, the tumor supressor gene BRCA1 is methylated and silenced, unlike in healthy cells where it’s expressed. The researches theorized that if we demethylated the BRCA1 gene promoter, we’d be able to increase expression of this tumor-supressing gene, and help stop cancer growth!

Demethlyation would be done by specialized proteins for the job, known as TET proteins. Direction of the protein complex to the BRCA1 gene region would be done by a guide RNA (sgRNA), and attaching both players together, would be our star: dCas9.

The Process

As we are only looking at the TET protein for its ability to demethylate, scientists only took the part of the TET protein responsible for doing that task, and attached it to dCas9. Acting as binder for both the guide RNA and the TET protein, dCas9 would be like a parent holding the hands of their two kids on either hand.

This entire family would be the “TDE” protein complex

This complex of TET, dCas9, and sgRNA all form into a protein complex called “TDE”. When TDE is infused into the cancer cell nucleus, it would travel to the BRCA1 gene promoter with guidance form its sgRNA, and then demethylate it.

Using this method, all members of the protein complex are fulfilling key parts in their demethylation task.

When discussing the consequences of enough demethylation in methylated BRCA1, the researchers predicted higher expression of cancer-suppressing traits such as tumor cell death (apoptosis) and lower cancer cell resiliency to chemotherapy drugs!

The Results and Our New Problems

When using a smaller TDE protein complex (TDE-I) alongside a specialized guide RNA (sgRNA2), the scientists achieved a relatively high rate of demethylation. This result was high enough to observe increased expression of the BRCA1 gene in cancerous cells! This over-expression of BRCA1 gene made the tested tumors overall weaker and less resilient, with our without chemotherapy exposure.

WOO! Does that mean we’ve cured cancer? … no.

There are still problems and kinks to be worked out, such as which specific parts of the BRCA1 gene should be demethylated for the maximum effect. Another large problem the scientists faced was that some demethylated areas got re-methylated (methylated again) by a protein called UHRF1, which is known to be cancer-causing.

Hurdles like UHRF1 and the need for more precised location epigenetic data for individual genes, is what we’re going to need to solve to make solutions like this work efficiently.

Key Takeaways

With this study laying down the idea of epigenetic manipulation to the most of our discretion, we’ve opened a bunch of new doors for ourselves in so many disciplines! The future potential of this relatively young epigenetics field is going to have a much greater technical impact on new healthcare solutions, longevity, and even human space exploration.

Some final takeaway points for this study and its implications:

  • Scientists proved that we can induce epigenetic manipulation in genes for our own purposes, given the right tools and attention to detail.
  • We have managed to weaken cancer cells across multiple human cell lines, by inducing artificial demethylation of a cancer suppressor gene.
  • In the process of inducing this up-regulation of BRCA1, we’ve also found out new roles that certain cancer-causing proteins are playing in cells. A prime example being UHRF1’s re-methylation of the demethylated BRCA1 promoter.

Epigenetics is undeniably complicated, and there’s an incredibly higher amount of questions than answers as new discoveries arise. This is a field in its infancy, with huge cross-industry applications to anywhere with biology involved. If we want to achieve some of our biggest biological ambitions, such as getting our species to interstellar space, or finding cures to cancer, it is only natural that the necessary work and complexity behind it will be huge.

Photo 51: Our first snapshot of DNA (crica 1952)

It’s been less than 20 years since epigenetics has taken off with significant progress in the field overall. From discovering that genes are being switched on/off, to discovering the heritability in epigenetic changes, and even now potentially looking at ways to reduce cancer; Epigenetics has come a long way!

As we continue to push forward here, everyday we bringing ourselves closer and closer to triumphing over our 200,000 year-old problems.

If you enjoyed this article, comment and give it a clap! I will be focusing on modelling some aspects of this research, so expect that in coming weeks.

Source Paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216816/