Gene Editing – CRISPR/Cas9

I saw this list of 13 female scientists who are doing major things in biotech in Silicon Valley and #1 is Dr. Jennifer Doudna, a faculty member at UC Berkeley who helped develop a technology you may have heard about: CRISPR/Cas-9. It’s the hot, new thing in biology research (for good reason) and could have implications for medicine as well (though they say that about every new biological and chemical tool and nothing ever works quite like we expect it to when we try working with humans).

So I wanted to explain a bit about what exactly it is beyond a “gene editing technique.” While I’m not using this tool right now, I had a related project in one of my rotations, and I may end up using it in the future (frogs have extra copies of chromosomes [humans have 2 of each] which makes genetic modification more difficult than in mice or fruit flies, but CRISPR/Cas9 works well and could be really useful for us frog people).

So what is it? First of all, in case you hear it spoken, it’s pronounced “Crisper Cass Nine”. The CRISPR part of it refers to the process (it stands for “Clustered Regularly-Interspersed Short Palindromic Repeats” but I actually had to google that because I didn’t know what it stood for, so not too important): it’s basically an immune system for bacteria (and archaea). Using this system, they can recognize foreign DNA and chop it up so it can’t do anything (such as be used to make proteins that infect or kill the cells). So CRISPR is the name of the process and Cas9 is the name of one of its effectors. Cas9 is the protein that does the chopping. It’s called a nuclease (nucle=related to nucleic acids like DNA and RNA and ase=it cuts something).

Now your cells are filled with nucleases. They’re used in all kinds of processes, including totally normal functions, but they’re tightly regulated so they can’t start chopping up your DNA (or to at least only chop it up when you want it to). So what makes Cas9 special is that it incorporates a piece of RNA that can specifically bind a certain sequence of DNA. If you remember from biology classes, DNA and RNA have bases that can pair with each other in certain ways. So As and Ts base pair and Cs and Gs base pair (see the adorable comic from Beatrice the Biologist below, whose name I just realized I totally ripped off, haha):

So Cas9 is a protein, but it can hold a piece of RNA that basically acts as an address. That piece of RNA can bind to the matching piece of DNA. And Cas9 will only cut when it’s RNA (called the “guide RNA”) matches the DNA (it doesn’t have to be a perfect match, but it has to be pretty close). And in its natural setting (bacteria and archaea), the guide RNA comes from foreign DNA that gets into the cell, so Cas9 targets this foreign DNA and chops it up. But in model systems in the lab, we can put in our own guide RNA.


It’s very easy to put DNA or RNA into cells. You can inject it (which is what you often do for frog embryos), you can electroporate it (electrically shocking the cells so that the cell membrane opens up and lets things outside the cell [such as the DNA you put in the media] in; this is used a lot with cell lines), you can introduce a virus (retroviruses will insert DNA sequences randomly into the genome). We’ve been able to do that for a long time. The problems were that either the DNA wouldn’t actually get incorporated into the genome (and DNA not in the genome sometimes doesn’t work as well as you’d like, and it won’t get copied and passed on if the cell divides), or it would get incorporated into a random part (which could disrupt other genes or cause other unintended problems).

But with CRISPR/Cas9 and a guide RNA for a specific sequence, you can make sure the DNA gets cut exactly (well, almost) where you want it to. Then the cell tries to correct that cut and it can do that in a couple of ways. It can try to just stick the two ends back together (which often leads to insertion or deletion of a few bases which can break the gene; this is called non-homologous end-joining). It can also try to repair the broken spot by using a template to remake what was damaged (this is called homologous recombination). And you can inject (or electroporate) a piece of DNA that provides a template that includes a new piece of DNA with whatever you want (for instance the blue “DNA” in the cartoon below).

This is one of the easiest and most effective ways we have of introducing a specific DNA sequence into a specific part of the genome, which is what makes it so exciting.

And from a medical standpoint, this could be really cool. A lot of diseases are caused by problems in single genes. If you can replace the bad gene with a good copy, the disease may go away (or at least not get worse). And with CRISPR/Cas9, you could potentially very faithfully insert a good copy in place of the old one (you can have two guide RNAs to cut on either side of the bad gene to remove the whole thing).

The first trick with doing this in humans is that you have to still get the guide RNA, the template DNA, and the Cas9 protein (or the DNA to encode it) into all (or at least many) of the person’s cells. And there isn’t a super great way to do that yet (viruses are the best, but they never seem to work as well as they “should”).

The second is arguably a much more difficult problem: ethics. One of the things that makes CRISPR/Cas9 so valuable for research is that it changes the DNA in the genome for the vast majority of the cells: including germ line cells. Germ line cells are the cells that become gametes, i.e. eggs and sperm. Which means that if you use CRISPR/Cas9 to treat someone, you will not only be changing their DNA, but the DNA of any children they may potentially have. Which doesn’t seem like a terrible idea for most heritable diseases, but we as a society have no idea where to draw the line. What even counts as a disease? That’s something that changes more than you might think. What’s the difference between a difference and disease when we define a lot of diseases (particularly neurological/social ones) as a difference from “normal”?* So until we can answer those basic questions, most people agree we probably shouldn’t mess too much with genome editing in people.

*I personally think the desert island test is a handy rule of thumb. If someone with a given condition would be perfectly happy alone on a desert island with nobody to treat them badly for being different, it’s probably just a difference. If they would be unhappy because the condition itself causes pain or suffering, then it might be a disease. Of course a lot of it is squishy and difficult and impossible to separate from society and culture.

I was just about to publish this post when I saw this article on CRISPR/Cas9 being used to modify (nonviable) human embryos. Talk about good timing. As the article mentioned, this is the second publication in which nonviable human embryos have been modified with CRISPR/Cas9. These two articles’ goals were proof-of-principle for therapeutic editing, so even though the embryos were nonviable and a product of normal IVF procedures, many scientists think these proof-of-principle studies should not be done until we’ve decided whether therapeutic editing is ethical (or under what conditions it’s ethical). As the article mentions, a UK group has also gotten approval (but not published results yet) to use CRISPR/Cas9 to study development in viable embryos. However the goal of the UK group is to study developmental processes that contribute to miscarriage (and their protocol has strict rules about the timeline of experiments so the embryos will get no further than a roughly spherical ball of cells about the size of a poppy seed), which is a fundamentally different goal. What do you think? Should we explore the use of CRISPR/Cas9 for explicitly medical purposes? Should it remain a research tool? Should it be used only for certain types of medicine? Only certain types of research? Plenty of existential questions to think about this weekend!


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