Friday, October 13, 2017

The First Attempt At Human CRISPR Gene Editing

The first attempt at human CRISPR gene editing did not occur in a hospital or University or in a clinical trial by some $100 million funded company. Instead, it happened in small cramped room in San Francisco in front of 30 or so people who squeezed in to listen to a talk about how biohackers are making genetic and cellular modification accessible.

This is the first time in the history of the Earth that humans are no longer slaves to the genetics they are born with. As I write this, the FDA is in the process of approving the first human gene therapy treatment. Still it's too slow for me, clinical trials have been going on since before 2008. I want to accelerate that. I want people to have a choice about their genetics. 

To push it forward I did a CRISPR experiment on myself.

How did I do this? CRISPR is some complicated new technology that is really hard! Well, at least that is what the press and media and maybe even some Scientists say. Unfortunately, it's not true. It just took one piece of DNA that contains the Cas9 protein and a guideRNA(gRNA) targeted to the exon 1 of the Myostatin gene and I injected my muscle with it. This DNA then enters some of my cells and both the Cas9 protein and the gRNA would be made by my cells and this molecular complex would target my myostatin gene and cut it. This would lead to Non-Homologous End Joining(NHEJ) and effectively some of the myostatin copies would not work. When myostatin is not working to stop muscle growth, muscles grow.  

Myostatin knock-out dog on left, normal dog on right.
Image from

I won't look like that dog though. The DNA only entered the area of injection in my forearm. However, the point of this experiment is not whether the CRISPR gene editing changed my muscles(too early to tell). Or that the efficiency was 100% and better than anything available. The point is that we are on the cusp of  humanity changing. This is the first of many people who will change their genomes. This will happen for medical reason, for science, athletics or maybe just because people wanted to or were bored.

I am sure many people will try and dismiss this as unscientific or how clinical trials will need to be run. The problem is that old world is dead. It died long ago and biohackers are creating a new one in its place. This is a world where the only important outcome is the one intended by the user. The FDA is meant to allow the creation of mass market drugs that companies can profit off of. It was never meant to regulate the genetics of individuals.

What could be more of a human right than to be able to decide what genes create you? 

About The Experiment

The one thing about a technology is that it takes infrastructure to grow. If everyone needs to program an app to use an app you would have very few people using apps and that is the state of lots of biotech and all of genetic engineering at the moment.

Fortunately, because of the hype, CRISPR has reached the place of infrastructure threshold because of all the companies that have been getting investments. What has happened is that lots of companies have been building up supporting technology to sell to the companies with lots of money. This means that the technology becomes more accessible to everyone. 

This is even the case for things like using CRISPR on humans. So why aren't people doing it or trying it? Are they scared? Don't want to? Or just don't understand how easy it is? Seriously, it is so easy that someone with little or no biotech knowledge could order DNA that contains Cas9 and a gRNA(a complete CRISPR system) and use it on themselves if they wished for under $400.

I'll be honest here. CRISPR modification is a bit overboard. In 99% of cases CRISPR modification is probably the wrong thing to do. Transient transfection has virtually no hazards and can lead to the same outcome. Still CRISPR is a fascinating technology. It blows my mind that in 2017 the technology to modify your genome is so easy to use. So I set about to use it.

One of the most common genes to target for proof of concept CRISPR or genetic engineering experiments is myostatin. Myostatin inhibits muscle growth so when you knock-out the gene it makes things have more muscle and become physically stronger. Examples( A knock-out generally just refers to a mutation that in some way causes the gene to not be made properly. In some genetic engineering methods you remove most of the gene by literally having it cut-out and replaced, this is where the term knock-out stems from. In CRISPR a knock-out from NHEJ often occurs from missense mutations that shift the whole DNA coding of the protein to make gibberish and so the gene doesn't work.

What about doing it on adults? There have been a few CRISPR experiments on non-embryos such as but most experiments with CRISPR are on embryos because then you can edit every cell easily as there are one or very few. This doesn't mean it doesn't work on adults just that you are working with percentages of cells rather than all the cells in the organism. 

Methods of getting DNA into cells can be as simple as putting the DNA in water/PBS and injecting it. The efficiency of this however is pretty low and is probably in the < 26% range of DNA getting into a cell. Still with billions of DNA molecules per injection this can be a decent number of cells. This technique of injecting DNA with PBS has been used in Phase I clinical trials( 

So has myostatin ever been edited or changed in an adult? There have been experiments on using DNA containing follistatin(follistatin is a myostatin inhibitor which stops myostatin from working instead of messing with the gene) and this has shown muscle growth in mice and macaques(non-human primates ( It can take months to see a difference in muscle growth though. Still this is not CRISPR.

CRISPR generally has lower efficiency than just the efficiency of getting the DNA into the cells because once in the cell the editing needs to take place and in the case of NHEJ a mutation needs to be made. So in order to make my injection more robust I mixed the DNA with PEI(polyethylenimine). This molecule has long been known to get DNA into cells more efficiently when injected or mixed with cells in culture ( 

The question of whether gene engineering will occur by injecting DNA with Cas9 and gRNA is mostly a non-question. There is an extremely high probability that the CRISPR DNA entered my cells and that some were edited. The biggest question is how many cells were edited and were there enough to have any physical effect? Even after the experiment I don't know. As only time and more experiments will tell.

A big question people have is, "What about off-target effects?"
According to a recent battle of the CRISPR off-target effects. Even using a gRNA that has predicted off-target sites in two mice led to 12 Single Nucleotide changes and 16 InDels(insertions or deletions) total among them.

In comparison in a human cell there is estimated to be anywhere from ~40,000 to ~150,000 DNA lesions caused by oxidative DNA damage at any given time. Cells also undergo ~50 double strand breaks per cell per cell cycle 

This is not to say that increasing double strand breaks(DNA being cut) is in any way helpful but more that human cells are very robust. Especially when the double strand breaks from CRISPR are targeted and are non-random meaning that through proper choice they chance that the can lead to a mutation that causes cancer or similar is even smaller than naturally occurring breaks.

To this day there has been no known occurrence of CRISPR causing spontaneous cancers to form.

I think it is ok to be fearful. But as a Scientist I think it is also a goal to look at fear and see if it is reasonably backed by numbers and understanding and compare that to the risk you are willing to take. You have about 5-10 trillion non-red blood cells in your body undergoing at least 50 double strand breaks every few days (not counting red blood cells because they make up a large portion of the bodies total cells and are boring). I can't imagine a few billion extra targeted double strand breaks would add much to risk. 10 billion extra double strand breaks(200 million cells at 50 double strand breaks from CRISPR(overestimate)) would amount to 0.0001% of the total going on in the body in the period of ~ a week. Over the course of a lifetime it would be infinitesimally small to be exposed to one CRISPR editing dose.

So I decided to go for it. I ordered the DNA online. There are many companies and websites that do this and so I will not mention the name of the company because I don't want to make any problems for myself or others.

Anyone can order DNA and can find a gRNA as long as they know the name of a gene(try it out with MSTN or myostatin). You can get a little more technical like I did and decide where you want to target and type of Cas9 used and other things but generally even if you had no idea really what you were doing you could come out on top.

Instead of having someone replicate the DNA to make lots for the injection(you can pay companies <$200) I grew up the bacteria that contained the CRISPR DNA myself and purified it. I basically did a double purification to make sure it was pretty pure(endotoxin free!). I used gel electrophoresis to assess the quality further. I then prepared 40ug of DNA with PEI in 350uL and it was ready for injection. 

The DNA sequence for the DNA I used can be found at:

After the injection there has been no swelling and only mild tenderness from plunging the needle into my muscle. The tenderness went away within two days of the injection. I am measuring both relaxed and flexed muscle circumference around the forearm where the site of the injection was.

The way to test to see how many cells were modified would require some "deep sequencing" i.e. I would need to do a muscle tissue biopsy and would need to sequence the myostatin gene in thousands of my muscle cells. I have contacted some companies for quotes.

Though this experiment was on me. I don't want it to be only about me. I want it to be about how this technology is inexpensive and easy to use. I want to it to be about helping people use this technology to better their lives and so I wrote a guide that shows people the steps of how easy it is to make your own CRISPR system.

The guide I wrote is meant to be general. Not meant to be in depth and weigh down people with details as I think genetic engineering shouldn't only be for people who can program DNA from scratch. Especially with the state of modern technology you don't need to and shouldn't need to know how to create gRNAs from scratch or their orientation &c. &c.

For people interested The CRISPR DNA is also listed on The ODIN but less for making money off of it(we are selling it dirt cheap) and more for making it accessible. Understand, the DNA we sell is not injectable. Once people have the DNA they can replicate it indefinitely in bacteria and grow up as much as they want and share it. It requires groups of people to make Scientific progress and effect societal change. I can't be the only one doing this stuff!

One of the main reasons I did this is to push the field of genetic engineering forward. Everyone wants to just talk about this stuff. They ask should we do it? I say, how can we not do it when we have a technology that can help people. 

This is the second time I have genetically modified myself. You can hear about the first successful attempt at