Editing the Genome of E.coli with CRISPR At Home
From theory to practice, how anyone can use CRISPR from anywhere to learn about gene editing.
Abstract
After spending months reading research papers and writing my own articles about gene editing, I wanted to take the theory and put it into practice. My first thought was that I need to belong to a prestigious university to even get access to a gene-editing technology such as CRISPR. But with a bit of internet scouring and guidance from a CRISPR researcher, I found The Odin, a biohacking company that sells educational CRISPR kits to anyone who wants to gain experience in gene editing. With The Odin’s gene-editing kit, I edited the genome of E. Coli with CRISPR to render them resistant to the antibiotic streptomycin. This experiment allowed me to develop my molecular biology and genetic engineering skills while exploring the current and future potentials of CRISPR technology, its legal and ethical concerns, and the importance of biohacking in instilling the responsible practice of gene editing by giving students and the masses access to the technology.
Video of experiment step-by-step: https://www.youtube.com/watch?v=FJrZG_zuVuA
Introduction
What is biohacking?
The experiment I conducted at home was a form of biohacking, a novel type of DIY biology. biohacking is a term that is used to refer to a wide range of activities that use biological concepts outside of the lab. For example, Intermittent fasting and specific dietary regimens are types of biohacking. But more experimental types of biohacking are similar to the at-home experiment that I did, where genetic engineering technology is used to edit the genome of bacteria. Essentially, most types of biohacking “hack” the biology of a living organism to enhance its bodily performance. There are many legal and ethical concerns when it comes to biohacking, but as the practice of biohacking becomes more responsible, we are likely to see more people hacking their biology in the future.
What is The Odin?
The people biohacking themselves or microscopic organisms are scientists with a Ph.D., enthusiastic students who want to get access to high-tech technology that is otherwise unattainable, and non-professionals who want to experiment with biology. A well-known biohacker who also has scientific accreditation to his name is Josiah Zayner, who founded The Odin, a biohacking company. The Odin creates genetic engineering kits that allow anyone to manipulate the genome of organisms at home or in a lab or anywhere (except not on human beings). The Odin’s DIY bacterial gene engineering CRISPR kit has already prepared a Cas 9 enzyme, gRNA and a template DNA that create antibiotic-resistant bacteria. This biohacking experiment is the perfect opportunity to practice molecular biology and genetic engineering techniques.
Streptomycin: Prevention of Protein Secretion
The goal of this genetic engineering experiment is to make a genome mutation (K43T) to the rpsL gene, changing the 43rd amino acid Lysine (K) to a Threonine (T) which will allow the E.coli to survive on streptomycin media which would normally prevent the growth of the bacteria.
The body of living organisms makes proteins, long chains of amino acids, in order to function. Proteins are made by cellular devices called ribosomes. The two processes that lead to protein secretion are transcription and translation. The code for the amino acids is located on the DNA which is transcribed on an mRNA. This mRNA is small enough to exit the nucleus and attach itself to a ribosome in the cytoplasm. The ribosome translates codons of the mRNA code into amino acids carried over by tRNA. The amino acids chain together and form a protein.
In this experiment, the bacteria is being grown on a media that contains molecules of streptomycin which attaches to the ribosome and prevents it from making proteins. Consequently, the bacteria cannot produce proteins necessary for their survival, replication and growth.
CRISPR: Preventing Streptomycin Binding to Ribosomes
In order to prevent the streptomycin from attaching to the ribosomes, a mutation is made in the ribosomal subunit protein rpsL which will secrete a protein, preventing streptomycin from binding to the ribosome. Thus, allowing the bacteria to grow on the streptomycin media. Essentially, a single DNA base is being changed so that the lysine amino acid at position 43 is turned to threonine.
This genetic edit will be made using a CRISPR-Cas9 complex (a gRNA and a Cas9 enzyme) and a template DNA which will make the substitution mutation.
I explain in detail how CRISPR-Cas9 is used for gene editing in this article: https://dibadin.medium.com/crispr-101-everything-you-need-to-know-about-crispr-970e58cc4b27
Materials
Non-perishables
- LB agar media: used to culture a colony of bacteria.
- LB strep ken arabinose: used to test if the edited bacteria can survive in streptomycin.
- Tube for measuring LB media
- 250mL glass bottle: used to measure water for LB media.
- A 100 µl pipette: used to measure bacteria, water, gRNA, Cas9, DNA template, LB media.
- Pipette tips: change between each use.
- Microcentrifuge tubes: used to store experimental bacteria.
- Microcentrifuge tube rack: used to store the microcentrifuge tubes.
- Petri dishes: to culture bacteria in LB agar media and conduct the experiment on LB Strep ken arabinose.
- Gloves: a safety precaution because bacteria is handled.
- Microcentrifuge tubes containing LB broth: LB broth is used to culture the transfected bacteria.
- Bacterial transformation buffer.
- Inoculation loops: used to spread the bacteria on the petri dish.
Perishables
(should be stored in a freezer upon arrival)
- gRNA plasmid: guides Cas9 to the target sequence.
- Cas9 plasmid: makes a double-stranded break on the DNA.
- Template DNA: inserts a single DNA base to make a substitution.
- Non-pathogenic E.coli.
- Sterile water tube: to extract the gRNA, Cas9, DNA template, and E.coli from their containers.
Methodology
Preparatory steps
LB agar and LB Strep ken arabinose plates were prepared. The LB media was mixed with water, melted in the microwave and poured into an equal number of Petri dishes.
Culturing the bacteria
The bacteria were taken out of its tube with sterile water using a pipette and then put in the corner of a petri dish. The bacteria were spread out in a zig-zag pattern on the LB media using an inoculation loop. The bacteria were grown at 23º C for two days. By the end of the two days, a cloudy-looking layer started to appear on the petri dish: this is the bacteria colony.
Preparing bacteria for transfection
The bacterial transformation buffer is put into a microcentrifuge tube with a pipette. An inoculation loop is used to scoop out a small number of bacteria which is then mixed into the transformation buffer. The resulting solution should be cloudy-looking.
I made 3 experimental samples and used 2 for the final results.
The samples were kept in the fridge at 4ºC for one day. The remainder of the bacteria were stored at room temperature.
Applying the gRNA plasmid, Cas9 plasmid and DNA template plasmid
Each plasmid was taken out of its tube with sterile water and a pipette and put into the samples. The microcentrifuge containing the sample was shaken and tapped against a table to get the DNA to the bottom of the microcentrifuge tube. After storing the samples for 30 min in a 4ºC fridge, the samples were heat-shocked by placing them in 42ºC water for 30 seconds. The bacteria were heat-shocked because the pore membranes had to be opened up, allowing the plasmids to enter into the bacteria. The temperature of the water was approximated by hand. For more accuracy, a thermometer should be used.
The LB broth contained in a microcentrifuge was mixed with water and mixed with the sample. The microcentrifuge containing the LB Broth and the sample were then shaken to mix the two solutions.
The samples were incubated at room temperature (make sure not at 37ºC!) to allow the DNA to replicate itself. One sample was incubated for one day, the other was incubated for two days. The samples were not identified.
The samples are put on the LB strep ken arabinose plates and stored at room temperature (make sure not at 37ºC!) for 13 days.
Results
Some bacteria were able to grow on the streptomycin media, as seen by the small white/yellowish spots that started to appear on the plate. However, the bacteria did not grow much in the first week; a few white/yellowish spots appeared on the plate. After 13 days, most spots on the plate became brown because the colonies were growing old. The remaining white spots were still young colonies. Consequently, some E.coli were able to survive on streptomycin media, suggesting that the CRISPR experiment did work. Further tests need to be done such as DNA sequencing to find out if the CRISPR was able to make the desired mutation or if the survival of the bacteria is due to a random mutation. I did not have the materials at home to conduct further analysis and tests on the samples.
Discussion
Odin’s DIY bacterial gene engineering CRISPR is a simple yet effective representation of the power of gene editing. This experiment was focused on making antibiotic-resistant bacteria with CRISPR, nevertheless, CRISPR and other gene-editing technologies can be used for numerous other applications. CRISPR has a wide array of applications in biohacking, agriculture, genetic diseases, cancer treatment, climate change solutions, etc.
While the positive applications of gene editing are plentiful, legal and ethical concerns such as bioterrorism and designer babies should first be addressed. Irresponsible use of gene editing can have negative consequences in human and natural ecosystems. While biohacking companies such as Odin are making the purchase and use of CRISPR widely available, they design their kits in accordance with bio-safety standards and non-hazardous material.
Nevertheless, with an education science kit like the Odin’s, students who are passionate about gene editing and the future of biotechnology, can start learning the fundamental techniques and concepts of molecular biology and genetic engineering. In conclusion, making gene editing technologies available to students responsibly through biohacking is an important step towards a future of ethical and responsible gene editing.
To learn more about biohacking: https://www.vox.com/future-perfect/2019/6/25/18682583/biohacking-transhumanism-human-augmentation-genetic-engineering-crispr
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