Dragon Creation: Using CRISPR to make real-life Dragons 🐲
A hypothetical CRISPR-cas9 guide for new species.
I would like to welcome you to the course “Dragon Creation: Using CRISPR to make real-life Dragons.” As many of you may know, the Komodo Dragon is a large lizard native to Indonesia. It is said that these reptiles are dangerous and predatory. However, with the right genetic modification… WE CAN MAKE DRAGONS!
Image from Britannica. Check them out afterward if you want to learn more about Komodo dragons!
A dragon is a mythical lizard-like beast with wings, it is said to breathe fire and is intelligent and cunning. This is a course about creating Dragons from Komodo Dragons with CRISPR so they can be our friends or scientific study creatures, maybe even pets 🐶 and NOT a gladiator creature for our amusement 😅
In this course you will learn:
The basic 15 steps of CRISPR-cas9 for this experiment.
How to engineer a dragon from a Komodo Dragon using CRISPR-cas9.
Possibly illegal gene splicing ‘ideas’ 🤐
Some of the tools needed 🔑
-Computer to map/plan out DNA strands.
-DNA extraction kit. Recommend CRISPR-cas9 as this course is all about that.
-Gel to identify the edited gene.
-Electroporator to carry out electroshocks to get Cas9 into the cell.
-Pipette for pipetting (of Cas9, RNA, DNA).
-A source for gene sequences like NCBI.
-PCR machine.
-A Freezer for the cas9 protein after it’s purified.
-Liquid nitrogen storage to store cell cultures.
-Cell Centrifuge for separation of growth media.
To get started- CRISPR:
Step 1: Purify the Cas9 protein. I recommend using a peristaltic pump: purifying it until you get a pure Cas9 protein. Go through it at least thrice to ensure purity. Have patience as this is a relatively lengthy process ☺
Picture of a peristaltic pump.
Step 2: Once you are finished the purification- Store the purified Cas9 in a freezer for the later gene editing experiment.
Image Credit: B Medical Systems S.à r.l
Step 3: Obtain the necessary cell cultures for the experiment. Use liquid nitrogen storage to store said cell cultures. Cell cultures needed are listed under the ‘Dragon Creation’ section further below.
Illustration of liquid nitrogen storage.
Step 4: Lift the cells from the plate and then electroporate them with an electric charge. We do this so that the Cas9 can get into the cell and go into the nucleus, hence editing the cells. Check out this article for more information on electroporation.
Step 5: Prepare growth media and cell cultures. Warm up the cell culture media to 37 degrees. You can get the media here.
Step 5,5: Clean the benches and workplace you are going to perform this experiment to increase the odds of getting uncontaminated products. Use wipes and gloves always. Also, spray the outside of the flasks when you take them out of the preparation station.
Step 6: Suspend the cells in the media and incubate them to promote growth. I always recommend using a shaker table as this allows the cells to mix in with the media perfectly. Also, the temperature should be kept at 37 degrees (or 98.6 F). The shaker tables usually are set to 8 hours. That usually gives enough time for cell cultures to expand and be big enough to do the CRISPR. If you want to check on the cells, you can inspect them with a microscope.
Step 7: Spin down the cells and remove the media. Use a centrifuge which is essentially a large machine that uses centrifugal forces to separate the cell from the media. The centrifuge will then give you a pellet, which is the cells you were looking for. Then what you can do is aspirate the media to the last drop.
Image of a centrifuge by hettweb.
Step 8: Cas9 has two binding sites which two non-coding RNA strands can associate with. Cas9 has a 'cleavage site' where it can cut the DNA strand. The RNA strand tells the Cas9 where to cut. Thus, you get the 'guide RNA'. You can get them both from the NCBI website; you have to order them together. Combine the Cas9 protein and RNA to form a DNA-cleaving RNP complex.
Step 9: Now you need the pipette for the Cas9 RNP to combine the cell cultures with RNP.
Step 10: Then we electroporate the cells to increase membrane permeability and facilitate entry of RNP.
Step 11: Incubate cells to allow RNP to perform DNA editing. In a CO2-enriched environment (e.g. 5 or 6%), using a CO2 sensor. Make sure the incubator is set to 37 degrees. If the cells have Cas9+RNA, they will be edited after 24+ hours of incubation. Here are the conditions for incubation: you should keep the cells in a dark place. For this experiment keep the cells in the incubator for 36-48 hours to allow the Cas9/RNP to perform DNA editing later on.
Photo-Step 12: It is vital that, after editing a gene, we take before photos so we can see a difference visually between a cell with Cas9 gene editing and an unedited cell. Also, we take before and after photos on a gel to see if the gene worked (i.e. DNA fragment is removed). And it is good practice too!
Step 13: Extract DNA and check on a gel before sequencing.
Step 15: Analyse gel and final DNA sequence to confirm successful CRISPR edit. Use a PCR machine to perform this step.
— Finished —
To the editing: 🧬
So, now you know the ‘how-to’, here is the ‘what-to’ for more concrete gene expressions and editing:
In this experiment, we will use a Komodo dragon as the basis for making a dragon 🦎
Step 1: To create a dragon, the first step would be to use mRNA encoding for a membrane protein called ‘sonic hedgehog’. Sonic hedgehog (or SHH as it is often known) is an endogenous gene that influences limb development during embryogenesis.
Step 2: Now we need to give it Hollow bones to enable flight in conjunction with later steps. Hollow bones are less strong than solid bones, but they make the dragon more nimble in flight. Great eagle hollow bone formation involves 2 transcription factors: HOPX and HOXD11, which will influence both bone length and bone diameter.
Step 3: To increase the size of the komodo dragon, you need to first modify the DNA. Specifically, you need to induce the overproduction of GH (growth hormone) by manipulating the DNA of the pituitary gland. To create elephant-like hollow bones, you need to induce overproduction of the hormones PTH (parathyroid hormone) and PTHrX (parathyroid hormone-related protein). In the case of GH, you need to use the Cre-Lox system to genetically modify the pituitary gland, where GH is produced. In simple terms, you use the Cre recombinase enzyme and insert it into the pituitary gland DNA. You then crossbreed it and see if it is passed down.
Step 3 explanation and further clarifications: Crossbreeding is an in-vitro (or in-living) process where you insert the genetically modified sperm into an egg and create an embryo in a medium. We then analyze the embryos with tools like PCR, DNA sequencing, Sanger sequencing, and bioinformatics analysis to see how much of the trait has been passed down.
On average, crossbreeding takes about 2-3 months (as it depends on the complexity of the desired genetic characteristics, like the ones we are trying for.) We insert the embryo into a test tube and put it into a suitable medium, so it can perform its proper mitosis and develop.
A good medium for growing a komodo dragon embryo would be the following: Dulbecco's Modified Eagle's medium (DMEM), supplemented with 20 mg% fetal bovine serum and 3mg% calf serum.
The fetal bovine serum (FBS), aka. bovine serum is blood plasma taken from the fetus of cattle and calf serum (CS) is plasma taken from calves. They are the most commonly used blood plasma for animals.
The ‘%’ here stands for %vol/vol percent. It just means you mix in 20 percent fetal bovine serum and 3 percent calf serum in the medium (DMEM) that Komodo embryos will grow up in.
Step 3,5: You need about 50 mL of medium per Komodo embryo, as the medium will help them grow properly and perform mitosis. You can use either a Petri dish or a T-175 flask! To avoid contamination due to fungi and microorganisms, you need to replace the medium every 5 days. Make sure to disinfect your tools first before adding the replaced medium into the test tubes.
We do keep a constant temperature- You can set the incubator to 37-38.5°C, which is the perfect temperature for komodo dragon embryo development. You can’t use an artificial womb because you need to inject something into the embryo at a later date (around the 2nd month of development)- This is called the injection stage.
Once the embryo has developed into a fetus, you can choose to implant it inside a Komodo dragon and see if the Komodo dragon can give birth safely to the fetus.
You can even use a CO2 incubator in a lab to do that and it would be very much preferred. A CO2 incubator helps maintain more controlled environments inside the incubator- for example, you can reduce the amount of air circulation inside, thus greatly reducing the amount of contamination possible with microorganisms/bacteria that can spoil the experiment.
We use a petri dish to test if the genetic material you just gave the new sperm has been successfully passed down to the embryo itself, which is growing in the petri dish.
What we have so far: Ok, so you have now made a crossbred Komodo dragon embryo which is genetically modified to over-produce GH (growth hormone). You put it in an incubator and let it grow, and you are now ready for step 2 of the process, the induction of hollow bones (so that when we introduce wings later on it can fly effectively).
Hollow Bones and wings:
Now for the beginning of wing development, we need to know what wings we want. I’ll do a pair of strong big bat wings for the komodo to have, but you can choose anything else that fits- expect some trial and error.
Step 4: For bat wings, you require the sonic hedgehog (aka. SHH) gene product. To induce hollow bones, you require overproduction of the parathyroid hormone 1r(PTHrX) and PTH1.
PTH, PTH1r, and PTH5 are all parathyroids. Their function is the regulation of calcium levels in the blood. This means they also play a role in the development of bones.
You need to engineer the embryo in a way so that PTH genes are induced, creating PTHrX+PTH1r gene products in its body.
4,5- a further explanation: To engineer the embryo, you need to get a cre-lox recombinase to insert PTH genes and PTH1r+PTHrX gene products into Komodo's embryonic genome.
You do this with the help of PTH-gene probes, which carry cre-lox recombinase. You insert the probe into Komodo's genome and then use the cre-lox recombinase to induce the PTH genetics.
Use a guide RNA to create a DNA double-strand break and use cre-lox recombinase to insert a PTH1r+PTHrx gene product into his genome.
Step 5: Next step is to start making it larger- you need to use a gene that activates its growth hormone production, and use a somatotropin gene to make it larger.
You also need to use the Sox 4 and Sox 11 genes, which are known to increase bone density and size. You would engineer these genes in the same way as with hollow bones. What I mean is that you use the same technique as the hollow bone modification, so you use the cre-lox recombinase to insert the Sox 4 + Sox 11 gene product into the genome. If you combine this with the GH gene, and some time for growth- you get close to a dragon!
Fire breathing:
Upon successful tries of previous instructions, we can now get into the fiery depths of dragons, the classical fire breath 🔥
Step 6:You would need to create a genetically engineered komodo dragon with the pyruvate oxidase gene product. To do this, you need to use an E. coli culture, along with the pyruvate oxidase enzyme, to make a genetically engineered DNA vector containing the pyruvate oxidase gene.
You give the Komodo dragon embryo pyruvate oxidase, and the gene is induced- you would need to combine this with a gas gland, and have the Komodo dragon exhale fire! Well… easier said than done, but here it is (hypothetically):
Step 6,3-DNA vector for pyruvate oxidase gene: You need to create a gene vector containing the pyruvate oxidase gene, using a program like DNA sequence software.
You then copy that gene sequence into a vector using a restriction enzyme, then you place that vector into E-coli to reproduce it.
You then need to take the pyruvate oxidase gene from the genetically engineered E. coli and inject it inside the Komodo dragon embryo.
A good place to inject it would be into the egg yolk, and make sure you use a pipette for the injections. You need a high injection volume for this project so make sure you adjust the size of the pipette accordingly.
Step 6,6- the gas gland containment vectors: Here's how you do it. You engineer the Komodo dragon embryo so that the glands secrete hydrogen peroxide. You engineer another part of the Komodo dragon, called the gas glands, to secrete ethanol.
This is important because hydrogen peroxide and ethanol will combine to make ethanoic acid. Ethanoic acid is a liquid, so it can be stored in the gas glands.
You first need to obtain the two gene products- hydrogen peroxide and ethanol. These are both enzymes, and so to obtain them you need to create a gene vector that has the gene sequence for hydrogen peroxide and ethanol and add that to an E-coli bacteria to make a culture.
Once you have a high enough amount of hydrogen peroxide and ethanol, you inject this into a Komodo dragon embryo which already has the gas glands in production.
Step 6,9- the gas gland: You use a Cre-lox recombinase to insert the following: 2 gene products- SPP1, and DMP-1.
These will both induce the production of the gas glands in a Komodo dragon, along with the expression of an enzyme called dentine matrix protein 1
Why?
The dentine matrix protein 1 (DMP-1) will induce bone formation and increase the bone size and bone density, while SPP1 will cause bone resorption and mineral formation.
The combined gene product from these two is what creates your Komodo dragon's new gas glands!
Step 7- Give it whale lungs: To increase the range and power of fire breath, consider this:
We need a bigger lung capacity to increase the range and power of the fire breath. I suggest taking the genes from whales to do this: GJA1, SCNN1A, and MUC12. Also, for the whale to have the ability to dive deep with those lungs, a gene known as KMT2D would be needed too.
GJA1 is needed for the connection of alveoli and blood vessels to increase the lung’s surface area.
SCNN1A is an ion channel to transport water through tissues.
MUC12 is needed to form respiratory mucus in the whale.
KMT2D is for the whale's heart to be more efficient.
Step 8: The genes we should get next, to improve lung and tissue functions are:
LRP5, to regulate the growth and structure of lungs.
FGF2, to regulate the cell metabolism in lung tissues.
PDZRN3, to allow cells to produce more mucus.
Step 9: To prevent possible lung cancers, we can use ABCA3 and NRF2. We could use VHL as it prevents the growth of blood vessels in the lungs, but I would not recommend it as it possibly will stunt the growth of the lung structures.
ABCA3 prevents inflammation after cell contact with environmental pollutants.
NRF2 removes harmful and carcinogenic agents from the lungs.
Step 10: Lung longevity is improved by FOXP3 (a gene-controlling immunity), KREM, and IL13. FoxP3 and KREM prevent inflammation of lung tissues and IL13 fights against asthma and allergies.
Step 11- An experimental choice: We can attempt to add:
TBX4, to help develop limbs in embryos.
HOXA13, to help coordinate the growth of bones in the limbs.
HGF has a similar role to HOXA13.
WNT7a also acts as a growth gene necessary for limb development in embryos.
Sight and seeing:
Step 12:
GJA3, this protein controls the flow of fluid and salt in the eye tissues.
RDH5, this gene is used to make retinol-binding proteins necessary for vision.
RPE65, this gene controls vitamin A for optimal vision.
CRB1, this gene controls the cell differentiation process in the retina.
Step 13: To have eagle vision, which is the best in the world, we can take help from eagle genes like:
TNR, this gene in the eagle gives it good vision at night.
BMP2, this gene allows the eagle to have a good field of view of 90%.
RBP1, this gene will improve the sensitivity to light and vision in dimly-lit areas.
Brain Development (optional)- Warning ❗ Will make the dragon highly intelligent (If done correctly):
To make the ‘dragon’ smarter, as dragons are often depicted as somewhat highly intelligent creatures, we need these genes as a fundament:
TBR1/TBR1l, are needed for brain development.
KCNJ2 and CHRNA4, these genes are good for brain signaling transmission.
KCNQ2 will help to improve overall brain and memory performance.
Now for the big ones that will truly make a change in the ‘dragon’ model, we are going to implement the following:
ARHGAP11b. It’s a human-specific DNA, and it works as far as we understand to promote the development of the cerebral cortex in humans. It should give the dragon model a larger brain and better thinking capabilities. It is also vital for the language processing part of the brain, called Wernicke's area and Broca's area.
SPOCK2 allows for more neurons and improves the efficiency of brain functioning.
CREM is needed for the formation of memory.
PRAME, allows the brain to develop sensory organs and other parts of the brain.
Now, you have a dragon :) If you want horns (which is kinda dangerous…) you can go through the following steps:
Horns (optional):
SHH creates the basic tissue for the horns to form.
HOXA13 & SOX2 to coordinate the growth of the horn tissues.
WNT10A to give the horn shape and size.
You may also need a protein called Bmp2 for the growth of the horn.
The BMP2 and SOX2 genes are activated by the SHH gene.
BMP4 to make an efficient and “good-looking” horn.
PTH1R activates the BMP4 gene and gives it a more efficient horn.
Now, you just need to find a set of horns you want, I’ll choose goat horns:
RHOA, this gene is useful in the growth of horns.
BMP4, for horn growth.
BMP5, for horn growth. This is for cosmetic purposes as this gene gives the goat its iconic curve in horns. -Also, this gene is also responsible for the goat's skull shape.
WNT7A, for horn growth.
Tha-da, done, now you have your dragon 🙌🥳 Have fun and be careful.
🐲 Disclaimer: This is all hypothetical, so use this information with caution. I am not liable for anything going wrong, such as being eaten.
For todays question to solve: How do we edit a our dragon to be fire resistant? A tip: You would need to isolate the trait of fire immunity/ fire resistance. There are many ways to do this, primarily by selecting species that already have immunity/ great resitances to fire.
Let's first make it clear, by "Fire-resistant I mean the "dragon's" scales. Here's another hint: The scales of the komodo dragons and related to that of birds. They contain an organic protein called β-keratin. The cells that produce β-keratin are called keratocytes.
To have it fire resistant, we would need to make the β-keratin to be heat resistant, or add a coating of some kind 🤔 Ok, good luck! :)