How to Genetically Engineer a Human in Your Garage - Part II - DNA got what I need

Ohhh babbbyyy, you, you got what I need


To embark on an experiment to start genetically modifying myself I needed to obtain DNA and not just any DNA, some that was specifically encoded to work with humans. This is because not all DNA works with every organism. In fact most organism have their own unique codes that allow and don't allow certain DNA to work with them. Kind of like a security system for the cell. Like the CRISPR kits we sell won't work on humans without modification, they only work on bacteria.

The purpose of most DNA used for genetic engineering is to make proteins¹ The targets I wanted to start with were Green Fluorescent Protein(GFP) and Red Fluorescent Protein(RFP). These are genes from Jellyfishes. I figured that in a best case scenario I would be able to actually see my cells glow and fluoresce if they were engineered. Worst case scenario is that I would need to extract the mRNA to look for the presence of these new genes in my cells but they would be easy to find if works because they are not endogenous(natural) to human beings.

Anyways, good thing for me there has been a bunch of work on modifying human cells with fluorescent proteins in petri dishes or cell culture as it is called. What this means is that there is already a bunch of DNA in existence that was made to work with human cells I just needed to find it and obtain it. Which can be more difficult than you think.

I am not going to out any companies specifically in this post because they may or may not want to know they are or have sent their DNA to me for the purpose of human genetic engineering. I am sure you can ask around or do research online and find companies that will sell to you.

I set about obtaining pieces of plasmid DNA^2,3.
And scary but not scary I wanted to obtain a virus⁴ that could put this jellyfish DNA into my cells.

One of the most difficult things about being a Biohacker is that companies sometimes won't sell things to you. Not because there are any laws regarding the sales but because they want to control who is able to do Science. Fuck that shit.


How to Obtain DNA
This is a best practices in how to obtain things that companies try and keep from individuals because they think we are stupid. Some cases may take much less and some cases much more.

1. Start a business. What this basically means is register a domain name and go through the process of setting up an email address so you can send emails from that domain. It legitimizes you 100x more than a gmail.com email address and the whole thing costs like $12 on godaddy.


2. Get a Ph.D. j/k j/k you can always just add Ph.D. after your name in an email. No one checks or can check. There is no database of Ph.D.s


3. Have a FedEx account #. I am pretty sure these are free and giving someone a FedEx account number has gotten me around the "We don't ship to home addresses" a few times.


4. Social Engineer them. It is blatant discrimination that companies won't sell materials because they think us people outside of academia or industry are too incompetent to use them properly or might hurt ourselves. And don't try and tell me it is for safety when I can purchase a gun, knife or car sooo much easier and those can outright kill people. Shit bleach and kitchen cleaner are more hazardous than any chemical in my lab and I can purchase those in a grocery store. Seriously, things like DNA are unduly controlled and we, at The ODIN, hope to do something about it in 2017. 

One of the companies I used is one of the largest suppliers of plasmids. They actually will ship to most anyone and most any address as far as I can tell. They have even encouraged me to lie to them to bypass their own review system. #FunFact


Ok, so what is the difference between using plasmid DNA and a virus?

Current, technology makes using a virus cost and skill prohibitive. Though I could purchase the virus for $500 or so it came in a limited supply. The benefit however is that it should work out of the box. I would not need to do much to make it ready. Plasmids are inexpensive, they don't work as well in terms of human engineering as viruses do but once you have some you can replicate as much as you want indefinitely. You can give it to friends to replicate and use and they are easy to modify for new uses.

I wanted to explore multiple avenues with my goal being to eventually focus on plasmid DNA because I want to work with technology that is cheap and accessible.

At first I began working with plasmid DNA. Though it is somewhat easy to work with it requires that one know how to do bacterial cell culture, plasmid purification and maybe even ethanol precipitation. You need a centrifuge and lab grade pipettes and probably an incubator and shaker. While most of this stuff will only run someone a few hundred dollars it is still a hindrance. However, I started a document on best practices for plasmid purification for human genetic modification. What one can do however is send the plasmid DNA or bacteria with plasmid DNA to a company and have them just purify you up a bunch. Depending on how much you need this can run from $100-$400+
Making and purifying your own viruses while not challenging from a technical or safety sense it is challenging from an equipment and resources sense. One would need to be able to do human or mammalian cell culture and transfection and have materials to purify the virus. Doable but these techniques are far outside the scope of a garage lab at the moment. Maybe not so much in the near future though?

The DNA in the virus came prepackaged and ready to go and despite what people might think, AAV is considered Biosafety containment level 1 by most places(which is the least strict and is on the same level as Sodium Chloride or Agar i.e. it is safe).

Obvi, after I received my DNA it was time for the experiments to begin! 

Genetic engineering in humans usually occurs as intramuscular injections or systemic injections(i.e. into the blood stream. Both of these ways seemed silly as they made it difficult to perform any testing to see if the experiment worked. Muscle biopsy on myself? YIKES! I decided on topical skin and subcutaneous(under the skin) delivery of DNA! The crazy thing is that there hasn't been as much scientific literature and research as I expected on this topic. I did collect a sampling of papers however that I found interesting and you can find them here: https://drive.google.com/open?id=0B_R75gIJvkFUMkhrbUdZRzhscGc


July 5th 2016 was the first day I started experimenting on myself.






Footnotes

1. Proteins are awesome. They are little molecular machines inside cells that let cells do things. Read about them!

2. Plasmid DNA is small circular DNA that is separately from the host cells genomic DNA(compared to genomic DNA. Genomic DNA is the DNA that is transferred from a parent cell. The standard set of genes each cell comes with). Plasmid DNA is circular so that it can be replicated easy by organisms such as bacteria. This allows researchers to grow bacteria and extract the plasmid to use for other purposes.

3. Here is a link to the DNA sequences of the plasmids and virus I used and am using: https://drive.google.com/open?id=0B_R75gIJvkFUMkhrbUdZRzhscGc

4. The virus in question is a non-replicative(it doesn't replicate and reproduce) Adeno Associated Virus(AAV). It has been used a bunch on humans and shown to be super safe. Fortunately, I was actually also able to obtain AAV1 which is efficacious across a number of tissues including keratinocytes(which make skin).

How to Genetically Engineer a Human in Your Garage - Part I

My name is Josiah Paul Zayner. If my name sounds "religious" that's because it is, Josiah was a King of Judah in the Hebrew.Christian Bible, Paul was an apostle of Jesus. I have three brothers Zachariah(older) and Micah and Jedidiah(both younger).

I don't remember a time when my Mom and my Biological father were together. He was severely abusive. We were poor. We lived on a farm. It wasn't a huge farm like people might imagine but a small animal farm in rural Indiana. We collected eggs from our chickens to eat and drank dehydrated milk. I have never met anyone else who drank dehydrated milk. I don't remember much of my childhood because of the trauma from abuse. I only see small flashes like a nightmare one can still feel emotionally but barely recall what exactly happened. This includes the now infamous family story of discharging the the gun my Mom had in the car door for protection. My Mom's second husband was also abusive. I ran away from home twice. Living with computer hacker friends in Oklahoma and Texas after high school.

My Mom is an amazing human. I would not be where I am today without her and her willingness to fight for her family.

Me(on the right side) and My Brothers circa 1987

Growing up I was never particularly what one would consider smart or gifted. I have just always liked to build and create things. I was mostly an introvert and would often get lost in my head, I still do. In high school I was mostly a jock but navigated through most groups. It was in high school that I found out about computers. I found out that if I wanted to learn something I could just read and teach myself. 

I made a discovery today.  I found a computer.  Wait a second, this is
cool.  It does what I want it to.  If it makes a mistake, it's because I
screwed it up.  Not because it doesn't like me...
                Or feels threatened by me...
                Or thinks I'm a smart ass...
                Or doesn't like teaching and shouldn't be here...
        Damn kid.  All he does is play games.  They're all alike.

The Conscience of a Hacker

My high school teachers made fun of me because I would always be reading in class. "What are you going to be a book reviewer one day?" and then the class would laugh and I would go back to my reading. I was almost expelled from High School twice. The first time I punched a kid a bunch and he ended up in the hospital. They handcuffed me in class. I was arrested and suspended. The second was for writing a program called "HackTheSchool.exe" and putting it on the shared network drive so anyone could access it. The program was a joke, it didn't actually hack the school but teachers back then didn't have enough knowledge to read the code and be able to tell the difference. In lieu of expulsion I was suspended and banned from ever using a computer in the school that was connected to a network or the internet. They had a special computer in my programming class that was unplugged from any network just for me.

Me on the computer at home circa 1999 

I eventually became a "computer hacker" and was a member of Legions of the Undeground(LoU) a hacking group. While that story is long and for another time it is the foundation of why I started my company The ODIN and have invested heavily in Biohackers. During that time in the 1990s and early 2000s I saw kids like me who were awful at school, came from broken homes, who could code circles around and out technical 99% of the world. All of us were self-taught. Little know fact is that you can still find a piece of software I wrote back then on The ODIN website and people still download it. Nowadays all of these people have great jobs at tech companies and most have still never finished college.

When I was 19, I was able to obtain a job at Motorola doing programming and networking. I worked there at the end of the dotcom bubble and then during the burst. I was laid off with tens or hundreds of thousands of other people. The one thing I took away from Motorola came from Dennis Tsai, he told me "People can take away your job but they can never take away what is up here (pointing at his head)." I could have taken another Tech Job but instead decided to goto school and learn. Fill my head with information that no one could ever take away.

Because I did so poorly in high school, I started back at a community college for around 1.5 years before transferring. I ended up majoring in Plant Biology and obtaining a BA from Southern Illinois University and though I did much better than I did in high school I just wasn't good at standardized education. Still there was a minor bit of meritocracy left back then and my Master's Degree at Appalachian(la-chun) State led to being accepted to a Ph.D. program at the University of Chicago.

I owe a huge debt to Ece Karatan, Tobin Sosnick and Tao Pan who taught me in Graduate School. Without them I wouldn't be who and where I am. I also think they owe me an apology because it has taken me years to unteach myself the critical nature I was driven into. I was taught to question everything and be constantly vigilant and critical. While this taught me to be logical and rigorous I think it stole my creativity, it stole my ability to dream and believe. Graduate school was a beautiful and extremely painful experience. It's hard for me to recommend graduate school, I think it really depends on the individual.

After graduate school I was awarded a fellowship working as a Synthetic Biologist at NASA engineering bacteria to break down and recycle materials for long-term space travel or when we colonize other planets. That was an interesting life experience but if you really want to read about that you can check out previous blog posts Here, Here and Here. Let's just say I don't work well in the system. I don't do well with rules that don't make much sense or people who are more interested in improving their position than contributing. Fuck the system.

The two guys named Bob who ran the NASA Fellowship program and Me

My last day at NASA and no one is around because no one is ever around.
Jan. 5th 2016

2015 culminated with me leaving my fellowship at NASA because I was fed up with the system, with everything. Outside I saw so many people hungering to be genetic designers and at NASA people hired to do the same siting on their asses. I began to work at The ODIN full-time. 

My goal at The ODIN has been to give people access to resources and knowledge so that they can Biohack. I have seen what hackers can do and usually because of their different path in life have unique insights into problems and tend to be more creative than trained and pedigreed Scientists. 

If a poor kid like me can end up at NASA and now running their own company but never quite be at the top of their class, never scoring well on tests no matter how much effort I put in, something is wrong with the school system. I can program 3 or 4 languages pretty fluently, can do electronics and basic circuit design and have a Ph.D. in Biophysics but even in graduate school no matter how much I studied I would always end up with lower grades than my peers. The education system is broken. The social system is broken. Or maybe they are all work fine and it is just me who is broken.

That's why I fight.

I fight for access for everyone. But I fight fiercely for the broken among us. Those who need hope.

You deserve better.

In 2016, I began to fight hard. I explored microbiome modification for health and privacy and did a thorough Scientific analysis that you can read Here, Here and Here. I found that it was possible to successfully transplant a gut microbiome and it alleviated alot of suffering in my life. It hurt and was hard.

In Sept. I ran the first conference for Biohackers(http://biohacktheplanet.com), which I worked my ass off for and poured so much into. It was amazing and turned out so much better than I could have ever hoped.

But still there is so much more to be done. The system is fucked up and so many people who hunger for knowledge and the ability to help others can't.

I didn't grow up wanting to be a Scientist or Biohacker or Genetic Designer. I was too poor to dream of that possibility. But, I did grow up wanting to help people and this has lead me to a project that will be a culmination of my life's work so far, Genetically Modifying humans. Genetically modifying myself.

This is me. I am a Genetic Designer.

Sorry, this post wasn't about the actual human genetic engineering experiment but I feel I need to give my background to tell my story.

Let's start at the beginning

I want the world to be like I see it in my head.

In April 2015 I was sitting around thinking. I like to sit around and think. And I began to wonder how genetic engineering technologies would be used in the future. I imagined a world in which people sought out genetic designers on craigslist and made a speculative post there just out of pure curiosity. Well the post was found and ended up going viral.

 

This lead to me studying CRISPR in depth, developing the DIY CRISPR kit(for microogranisms not humans!) and starting to think about how human beings could easily engineer themselves not just for health and medicine but also for "fun".

Around this time, an argument I constantly heard against human genetic engineering is that it would make class divides even greater. The wealthy would be able to afford genetic designers while the poor would not. That is of course if no one ever worked on making it inexpensive and accessible. What if I did? What if the masses have access to genetic modification before the wealthy because we are more clever and actually willing to try it?

So how do we begin?

There are two main ways to genetically modify any cell, including human cells, 1) Edit the genome, which is like the operating system of cell and so as you can imagine this can be dangerous 2) Add in DNA that is separate from the genome and is non-permanent, this also known as plasmid DNA, extra-chromosomal DNA, episomal DNA, transient transfection or a DNA "vaccine".

At first I only thought about (1) because I imagined the project as purely theoretical. I studied and wrote-up a feasible protocol and wanted to post it online for people to see and maybe use. I dug deep and figured out the best place for insertion into the human genome seemed to be this H11 locus and that one could deliver a CRISPR package in two separate viruses(CRISPR system is too big for only one). Use CMV driven SaCas9 and U6 driven guideRNA in an AAV serotype (serotype dependent on the cell type as they have different proclivities) with a viral titer of 10^11 or greater. You can do injections or intravenous infusions.

The more I read the more I understood how this could actually work and I became more interested in trying to use CRISPR on myself. But that seemed drastic and kind of stupid to try and make permanent genome edits on myself.

What about (2) though? Maybe I could use all the stuff I learned about studying CRISPR and try to transiently and non-permanently modify my own cells. From what I read it would only last a few days or weeks at most. I started to work on this part about a year ago and since then have explored a number of different techniques to express non-human proteins(GFP, RFP and others) in my own cells in my own body. I have been documenting with video recordings that I want to share so that others can learn and experience what it is like to push the cutting edge of Science.

My main goal is to develop methods so that genetic modification of human cells can be done by anyone safe, inexpensively and of course successfully. I plan to release as much information as I can about DNA used, reason for choosing different things. I plan to release the DNA code for the plasmid I have designed and protocols and techniques online(probably mostly on this blog) all to anyone for free.

Because there is alot of footage I plan to release posts on weeklish schedule or so. Which will include data, outcomes, footage and commentary. Going to try and not give spoilers!

For now I created a teaser with some footage from the past year and I hope you go on this journey with me to create something beautiful.