Gene Editing with CRISPR-Cas9 — Why We Should be Excited, but Humble

How I Got Into This

I am just nearing the end of my Bachelor of Medical Sciences degree at the University of Western Ontario, and I love science. Recently, I have taken up the habit of reading scientific articles for leisure (to be honest, this was to replace my reddit habits). Here and there, I have seen lots of interesting research coming out, but one thing keeps popping up.

“CRISPR-Cas9”

CRISPR-Cas9 has garnered a lot of attention in the past few years. It is a very efficient, cost-effective, easy-to-use and widely applicable form of genetic engineering.

In 2014, CRISPR-Cas9 was already gaining a lot of attention in the scientific community.

In fact, this week CRISPR-Cas9 saw some publicity as a CRISPR edited mushroom was genetically modified to resist browning, and successfully got past US regulations. One big reason for this was that CRISPR does not involve the introduction of any foreign DNA (such as from bacteria), and instead is a way to modify the existing DNA. Partly because of this caveat, the modified mushroom product was approved.

So, what exactly is this CRISPR stuff?

The CRISPR-Cas9 Technique

As one might expect, the authors over at Nature do a much better job of explaining this than me, a simple Bachelor of Science, ever could. Here, I will explain a little bit about how this fascinating technology works.

So let’s get to it!

Origins of CRISPR-Cas9

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and the Cas part is a term used to describe CRISPR-associated genes. Both CRISPR and Cas genes are actually a major part of the adaptive immune system in some bacteria and archea (check this out for some info). In this context, these species are able to use this mechanism in order to eliminate potentially unwanted genetic material. It is a highly efficient mechanism that can be targeted very well to DNA sequences.

Bacteria can use CRISPR and its associated Cas genes to protect themselves against viral infection. Source article

So, based on the origins of CRISPR, scientists have started to take advantage of it to do a bit of their own modifying.

Using CRISPR-Cas9 in the lab

When you are looking at CRISPR-Cas9, there are two important components that allow it to work. First, the Cas9 enzyme functions as a pair of “molecular scissors”, allowing for the cutting of DNA. Secondly, this is all targeted by a small RNA molecule, which can direct the machinery to the sequence it needs to modify.

This second part, the targeting, is what seems to really make this something worth talking about. The best part of this overall system is simply the efficiency, ease-of-use, and turnover. As it turns out, genetics and gene editing can call for some expensive and time consuming work. CRISPR seems to be a way of addressing this.

Combined with continued improvements in the areas of specificity and targeting, this type of tool can be used to do some amazing things.

Fun with CRISPR-Cas9

Some modifications have been made to the CRISPR-Cas9 system in order to have some different outcomes. Teams have sought to “break” the Cas9 enzyme so that instead of making its own cuts, it simply blocks the action of other proteins. By blocking proteins needed for gene expression, they can “turn off” a gene, without modifying or cutting out DNA.

It gets more interesting, though. Binding this whole machinery to an activating protein allows you to turn on the gene instead. Even further, scientists have been working out how to activate genes as desired, by using light.

Here is a great summary of what I’m talking about (source in caption):

CRISPR-Cas9 has allowed for us to turn genes on or off, and control expression in more exciting ways. Source

So…

In short, we can do a few things here. Firstly, there is the original function of CRISPR-Cas9 which is to target specific sequences and cut out DNA. In addition, the targeting component of this system has been applied to other ideas such as turning genes on and off with various methods. The specificity, speed and efficiency of CRISPR-Cas9 is what really makes it interesting and potentially useful in a lot of applications.

Using CRISPR-Cas9 to Fight Disease

Gene editing definitely has a lot of uses. The fields of medicine, basic science research, and food production all could stand to benefit from something like CRISPR. Here’s a decent diagram that sums it up, from a paper in Cell

What I want to focus on is the use of CRISPR in human health and disease (the “medicine” part of that diagram). Sure, mushrooms that don’t brown is a cool concept, but I think this is even cooler.

A quick search on PubMed can find many papers using CRISPR to either study, model or combat disease. But, what I will do is discuss a few really interesting ones: HIV, cystic fibrosis, and hemophilia.

HIV Resistance

Ten days ago, an extremely cool paper was published in the Journal of Assisted Reproduction and Genetics. You can read a nice discussion of the paper here (yes I like Nature).

Now, I am playing this up a little bit, but allow me to explain. There is a naturally occurring genetic mutation in humans of a gene called CCR5 which has been shown to confer resistance to HIV infection. This is because the protein that this gene encodes for is a component of the cells that HIV preferentially targets. Previous research has shown that stem cell treatment with a mutated CCR5 gene may be used to cure HIV infection. As discussed in that paper, the mutant allele we are looking at here is “CCR5Δ32”, which means that a small part of the gene has been deleted.

A depiction of how CCR5 mutations may confer resistance against HIV. This protein is an important part of the virus-receptor interaction that is needed for viral entry into T cells. Source

Ok, so back to that paper. What they managed to do was introduce CCR5Δ32 mutations into human embryos (which were nonviable, originally intended for in vitro fertilization but deemed unable to be used). The paper did not actually do any tests with HIV to measure the effects, but was more of a proof-of-concept. There were a few problems that they found, mainly having to do with an inability to attain a “fully” modified organism. Some cells did not get the mutation and remained normal (a concept known as genetic mosaicism).

But still, damn! This is really, really exciting. Now, what really got my attention in that paper was the discussion. The authors are extremely cautious, perhaps even a little afraid, of us rushing into this. Even in the abstract, they state:

We advocate preventing any application of genome editing on the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.

I mean, I think you’ve got something pretty powerful if you need to include that in your abstract. I am really excited to see what comes of this.

Cystic Fibrosis

I remember a couple years ago I participated in a case competition where we had to think of a way to combat cystic fibrosis. I wish I knew about CRISPR at the time. (Edit: apparently we were told about this in the case. Oops)

Cystic fibrosis is a very damaging genetic disease which causes huge problems due to excess mucous accumulation in the lungs. The centre of attention here is a protein called CFTR (cystic fibrosis transmembrane conductance regulator). CFTR can be mutated, resulting in the production of sticky mucous, which accumulates.

How cystic fibrosis develops. Without a functioning CFTR ion channel, mucous can accumulate in the lungs, leading to major health problems. Source

CRISPR-Cas9 has been used in order to repair nonfuctional CFTR, allowing individuals to potentially express fully functional CFTR proteins. Once again, we notice that a gene responsible for disease may be edited in order to overcome a major sickness.

Hemophilia

Hemophilia is another genetic disorder, and a brutal one. This disease involves excessive bleeding, even following small wounds. Reduced clotting ability in the blood is the problem here. The gene responsible is the F8 gene, encoding for an important blood coagulation factor.

Hemophilia impairs our ability to stop bleeding following an injury. Source with more info.

Once again, CRISPR-Cas9 has been used in the lab to combat this disease. Mouse models containing an “otherwise lethal” mutation showed restored gene expression and functionality following CRISPR-Cas9 mediated therapy.

These are just some of the papers that I have come across in my own leisure. And as always, I need to include the disclaimer that I’m heavily paraphrasing and summarizing here. You will find that the authors themselves are (as they should be) much more reserved with their language.

That said, who doesn’t like getting excited?

There is a ton of research going on using CRISPR techniques. Not only can we think of ways to fight disease, but it can be highly useful in modelling and studying biological mechanisms. If you can turn a gene off, or alter it in some way, you can observe the outcomes and figure out if that gene is important.

That’s Why We Should Be Excited — Here’s Why We Should Be Humble

Here’s the thing. We haven’t perfected this at all. The HIV paper I cited is actually a perfect example of this. They do not know the extent of undesired effects the treatment may have (but are looking into that), and they were not able to get uniform expression.

So that is one issue, the scientific one. The other is ethical. Are we ready for genetically edited embryos? When, and where, will the first human with CRISPR edited DNA be born? There are a lot of factors here. This paper goes into some interesting discussion about it, but I like their map of different countries’ stances on gene editing:

As you can see, around the world there are a lot of different perspectives. Will this change? I really do think a lot of discussion and debate about this is going to start happening.

In addition, the authors of that HIV paper raise some interesting questions in their discussion. What will we consider to be the “norm” for the human genome? We are getting better and better at identifying tons of genetic alterations, some good and some bad, in the genome. What measures should be taken in order to draw the line on genome editing?

However, let’s not forget why we love science. When discussing awesome, emerging scientific technologies, I always think about this SMBC comic about antimatter:

Right now, I am interested in this stuff because it’s just so damn cool. But we do need to take a step back sometimes and think about what we’re doing. I’m all for anything that can help us fight disease, but I think an open and mature discussion needs to be had about how we are going to implement such powerful technologies.

For now, we can rest assured that our mushrooms will not be browning anytime soon. However, I encourage you to keep an eye on this emerging technology.

What do you think?

Jim Denstedt

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