CRISPR Now Used To Create/Correct A Genetic Disease In An Adult Mouse

in science •  last year

One of the drawbacks of the CRISPR/Cas9 genome editing technology is getting it into cells, you can't just give someone a pill or injection and expect this large bulky protein and that RNA targeting guide to localize themselves to your desired destination. For instance when researchers use this technology on embryos they inject the protein and guides directly into the cells of the embryo (as we discussed Here previously).

In a new article published March 16th 2017 in the journal Nature: Scientific Reports titled "Somatic genome editing with CRISPR/Cas9 generates and corrects a metabolic disease." The authors describe their work on this difficult process of gene editing in "somatic" aka adult cells. The study is performed in mice, and the results are quite interesting, so lets take a brief moment and dive into this work.

Background on CRISPR/Cas9 Genome Editing

I have just recently written on this topic Here for more detail I refer you to the section I wrote about in support of it there.

However Briefly:

Cas9 or CRISPR associated protein 9, is an enzyme which can chop up DNA at specific locations using a short segment of RNA called a guide. That RNA guide's bases are complementary to a specific region of DNA somewhere else, and when it pairs up, then and only (within reason, non specific cutting is possible but not prominent) then does the Cas9 protein cut. Researchers use this unique property to target specific cites in genes to allow for repairs to be made to a broken sequence.

Why Were The Researchers Interested In Using CRISPR Editing In Adult Mice?

The article focuses on some genes involved in metabolic processing. The authors state that most research to date on metabolic diseases has happened in mice, where genes that cause the disease can be knocked out or mutated and the mice grown in order to study the disease.

Researchers like to use the non wireless mice, since they have tails!

All of this happens at the embryonic stage where manipulating the genome is easier. However there are some genes, where if you get rid of them or change them, are lethal to embryonic development. So the adult disease state can not be studied. If only researchers could cause the changes after growth into an adult, you know once development finished. This is where CRISPR comes into play.

However as I mentioned earlier it's difficult to get the CRISPR editing materials into the adult cells.

Delivery of Cas9 was not addressed in this particular publication, having it present in the cells was accomplished through use of a previously generated lineage of mice that have had expression of Cas9 already added to the genome of their cells. [3]

In this article the researchers tackle part of the issue, delivery of the guide RNA.

So How Did They Do It?

By using a virus. One way researchers have gotten CRISPR/Cas9 and guide materials into cells before is to use an adenovirus as a carrier to infect the cells and put it in there. [4] However these experiments were just on cells and not in an actual organism where any cell can potentially be infected with the adenovirus.

What Were They Targeting?

They were targeting two proteins which, when deleted are known to cause accumulation of fatty deposits in the liver. One is called low density lipoprotein receptor or "Ldlr", and the other is called Apolipoprotein B or "Apob". In the liver large fatty complexes composed of tryglycerides as well as Apob are formed, these complexes are called VLDL or very low density lipoprotein. They are shuttled out of the liver into the blood stream where they are broken down into tryglycerides and low density lipoprotein aka LDL. Ldlr is then what is responsible for shutting the LDL out of blood vessels.

It is also known that mutation to the Ldlr gene results in very high cholesterol levels and development of atherosclerosis aka a build up of fatty plaques in the arteries.

Figure reproduced from [2]

They infected the mice with adenoviruses containing the respective CRISPR/Cas9 guides for the genes of these two proteins then fed the mice a high fat high cholesterol diet for 20 weeks and examined their liver tissue (scheme is in a, above). They first checked the mRNA levels ( the mRNA is what is used as the instructions for building the proteins) and found that when a mouse was infected with the adenovirus that is supposed to knock out only Ldlr, there is a reduction in Ldlr mRNA (red bar in the bar graphs), when the mouse is infected with adenoviruses that will provide guides for both genes, the mRNA levels are reduced for both (blue bar in the bar graphs). The green bar in the graphs is a control that shouldn't knock down either protein, and it doesn't.

What Happened In The Mice When The Genes Were Knocked Down?

Figure reproduced from [2]

So we can see from (A) that when the mice were fed a high fat diet, the amount of cholesterol (LDL) in their blood increased this is the green line (makes sense). When the Ldlr was knocked out (red line) the amount of LDL increased even more, and when both Ldlr and Apob were knocked out the cholesterol levels in the blood dropped. All of this makes sense based upon what these proteins are supposed to do. Figure (B) shows that they only see a drastic increase in LDL when the Ldlr is knocked out, which is in line with the previous figures data. (C) shows where most of the tryglycerides were located, for the normal mice (green) some were in VLDL and Some were in HDL, the livers were functioning properly. Finally (D) they show that they found that atherosclerosis only was observed in the mice with the Ldlr knocked down by the CRISPR/Cas9 technology.

What Does All Of This Mean?

It means that the delivery of the guides by the adenovirus works!

The researchers here successfully re-created the symptoms of a disease which results from mutation of Ldlr, and they did it with CRISPR/Cas9 editing technology and delivery of guides with an adenovirus.

To date there has never been a model created for this, because knocking out these genes is lethal to embryos. It was the editing in adult cells that allowed for this to work.

That however is not all they showed here. They also showed that when you knock out the Apob protein as well as the Ldlr, that the atherosclerosis goes away. This illustrates the power of the gene editing technologies use as a means to potentially treat a disease, as they were able to treat the atherosclerosis here by inducing a second change. Still note that this particular example is certainly not the best example of a change one would want to make, it is however a proof of concept. This is a nice step in the right direction to showing that targeted gene editing can be used to both induce a gene state as well as correct for one.

Now we just need to work out how to deliver the Cas9 at the same time, but for that... more work will be necessary.



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Images from figures in Nature: Scientific Reports articles are available for reuse under a Creative Commons license.

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this is some great progress in the field. as exciting and great as it sounds however, I also have this sneaking suspicion that this may also carry some less than honorable ramifications along for the ride (another pandora's box, if you will)...

but it's still awesome, nonetheless, I hope . lol


We all hope.


Hahhaha this also could be the factual basis for resident evil part 20 :)


Depends on whether the umbrella corporation is formed ;)


Just a matter of time if you ask me. Will be a blockbuster if you ask me.

Very interesting article. Thank you!


Thank you for reading!

Nice read, I thought your approach was really well crafted. Speaking as someone with no background in biosci, I took a lot from that.


Great! My writing is a big work in progress, but comments like this indicates to me that I am getting there. Hopefully I can continue to improve and help you and others continue to learn some new stuff. :D

I has a ways to go but this could revolutionize treatment of many diseases.


Agreed, it needs more time but things are looking to be on the right track. Thanks for reading.

Will they ever be used clinically ? I have heard some clinical trials were recruiting.


This still is not ready for clinical trials IMO. In this experiment they were still using transgenic mice, ones which artificially already expressed the Cas9 nuclease, they just used the viral vector to deliver the guide to tell it where to cut, and used it to edit non embryonic cells (cool, and an important step in the right direction). The next step would be to get both the nuclease and the guide deliverable, which should in theory be possible through the same mechanism they used here, but thats a whole different research article and a ton more work.

Will this ever be used clinically? I sort of suspect it will. Will trials be starting tomorrow, I don't think so. However maybe there are some people that are further along with this then I have yet read about, or perhaps that has even been published. Clinical trials with this technology would be a pretty huge deal so we may not know until they actually start.

This scares me. Tampering with genomes should be left to evolution. Steemers would win out there so it is fine by me :)


I disagree, too many diseases can be fixed this way, to not at least investigate.


Thank you very much. :)