An interview with Rebecca Li, Aarthi Vijayakumar and Emily Gleason on exploring CRISPR-Cas9 functionality on the ISS
Rebecca Li (RL) – is now studying Molecular, Cellular, and Developmental Biology at Yale. She works in Dr. Amy Bei’s lab on the functional analysis of the malaria vaccine candidate PfRipr.
Aarthi Vijayakumar (AV) – is now studying Molecular Biophysics and Biochemistry at Yale. She works in Dr. Joan Steitz’s lab on the role of long noncoding RNAs produced by readthrough transcription on the antiviral response.
Emily Gleason (EG), Ph.D. – is the Curriculum Specialist at miniPCR bio. She received her Ph.D. in Biology from Harvard University before pursuing her passion for science education as a member of the Curriculum Fellows Program at Harvard Medical School.
Wolfgang Nellen (WN) – was a professor for genetics at Kassel University and is now working for Science Bridge e.V. on the public outreach project “CRISPR-Whisper”.
The Genes in Space contest (https://www.genesinspace.org/) challenges middle and high school students in the United States to design DNA experiments for space. The winning student proposal is then adapted and performed on the International Space Station. Genes in Space was founded by Boeing and miniPCR bio with the aim of inspiring the next generation of scientists, but it has also lead to some ground breaking experiments in space like the CRISPR experiment that was recently published in PLOS ONE (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0253403).
WN: Welcome to our small interview meeting. Thanks to Rebecca and Aarthi for joining and thanks to Emily for setting up the contact! Let me directly start with you Emily: Genes in Space has already done several experiments on the ISS. Who had the idea to bring CRISPR out there?
EG: It was 100% the idea of a team of high school students. Genes in Space is a contest that challenges high school students to develop DNA experiments for space. Every year we get hundreds of applications from all across the US. We read them all and in the end we have five finalist teams that compete to bring their experiment to the ISS using the tools that we had already developed. Scanning through the 500 proposals I saw this one on the investigation of DNA damage by radiation and microgravity and I thought „wow – this is great!“. But I am not in charge of selecting the contest winner. Fortunately, others recognized what a great idea this was and our panel of judges choose this as the winning proposal.
WN: Rebecca and Aarthi, tell us a bit about your background. As far as I understand you were high school students back in 2018 when you submitted the proposal and you are both now studying at Yale. How did the idea evolve to bring CRISPR to the ISS? What was your motivation?
RL: At that time, we had the chance to work in different bio-labs at the University of Minnesota. I was for example in a lab working on cancer drugs and our team mate David was in a lab working on CRISPR. We thought that was cool and started reading papers and discussing various issues and we thought that radiation and DNA breaks and DNA repair in space were real important to study. After discussing various set-ups we decided that the entire workflow (from setting breaks to repair and analysis) had to be carried out in space under micro gravity conditions.
WN: Sorry for forgetting! We have to mention the other two members of your team – David Li and Michelle Sung – who unfortunately can’t join today.
AV: We discovered the Genes in Space contest online just a couple of weeks before the submission deadline. We decided to write down our idea and actually submitted the night before it was due. Then we almost forgot that we had submitted. And then, a month later we got the call that we were among the finalists. Only then we told our parents that we had submitted for this contest and that we should go to San Francisco to present our idea to a panel of judges. We were just like in shock! We then had to work real hard to prepare for the conference in July reading up on all of the relevant research: among them the NASA twin-studies, the studies on cancer risks for astronauts returning from space and others. We thought it was a good idea to contribute to this research by our CRISPR experiment.
RL: Unfortunately, I had scheduled a trip to China at the time of the conference and the tickets were so expensive that I could not even go to the conference and present.
WN: But fortunately you had your colleagues to do the job in San Francisco! I am quite impressed by your background! To know about double strand brakes by radiation in space is one thing, to connect this with CRISPR-Cas is something different. People usually think about curing diseases, making transgenic crops and maybe even optimizing humans by CRISPR-Cas. You are using CRISPR-Cas for a completely different application and that’s quite clever. Tell me a bit more about the details of the experiment.
AV: The main question was if DNA damage is repaired differently in space, if there are preferred pathways, if repair was more or less efficient, more or less error-prone. This could provide hints to potential drug targets to mitigate cancer risks.
We wanted to simulate the entire process from DNA damage i.e., double strand breaks to repair and then sequence across the break site and see if it was correct or not. The breaks were induced by CRISPR-Cas at a specific site, cells were allowed some time to repair, then the region was amplified with the miniPCR machine and sequenced by the minION sequencing device directly on the ISS.
WN: I understand that you had to do the CRISPR induced breaks and the repair on the ISS. But why did you do the sequencing there? This was repeated anyway back on earth.
EG: We wanted to show that all the consecutive steps of the entire experiment can be performed in space. This is important for other space missions e.g. to Mars where you cannot easily send back the samples for further analysis on earth. And yes, re-sequencing on earth was required to make sure that everything worked well on the station. We also did not know before if minION sequencing was deep enough in the space environment to answer the question.
WN: What have you learned from the experiment?
AV: First of all, the entire workflow can be done in space and CRISPR works in space! That opens up many possibilities!
We do not have enough data to draw any conclusions if DNA repair works differently up there – this would be follow-up work.
We have also learned how difficult science in space is and what limitations you have on the ISS. Originally we were thinking about more sophisticated experiments with e.g. organoids, but there is simply not (yet) the infrastructure available.
RL: We then had to think together with the NASA scientists, with miniPCR and Boing, which reagents could be taken to the station. What is safe on earth is by far not considered safe on the ISS. Essentially, the NASA scientists get tense about any reagent beyond water! Then we had to think about biosafety and biohazard regulations and finally we had to conclude that we could not do everything that we had planned.
EG: … and still, what they have done is probably one of the most complex biological experiments. And there were a lot of “firsts” that they accomplished.
AV: Yes, this was the first time CRISPR-Cas9 was done in space, the first time cells were transformed in space. Most importantly, it was the first time that the entire workflow from transformation to the molecular analysis was carried out in space.
EG: And it was the first time, cells were plated on agar plates! Things that are trivial on earth are a real challenge on the ISS! Astronaut Christine Koch had to pipet tiny droplets that attached to the plate by surface tension instead of directly spreading a 120µl quantity that would have floated around in the station! Christine is an excellent scientist but she had never done anything like this before. So together with the NASA scientists, the team went step by step through the entire experiment and wrote down detailed instructions for the astronauts.
WN: I noticed that transformation efficiency was very low. Can you say if this was due to space conditions or to other more trivial factors like e.g. handling the cells?
EG: Well, we were just happy to get enough colonies to do the experiment. It could well be that the cells were not optimal. They were going through a lot: being frozen, put in a rocket, transfer to the station – many things that may have had an impact. We also found out later that the Cas9 plasmids that we used are somewhat toxic to the cells and could decrease transformation rate. So we can improve on that.
WN: Do you know of any plans of a follow-up experiment? You are probably not going to do it. But since you are now space experts, what would you suggest for the next mission?
AV: We do not know of any follow-up. But we hope that this research will be continued to answer our initial question. First of all, the experiment has to be repeated and expanded with better transformation efficiency to get more samples. And finally know better about repair in space and the implications on astronaut health.
WN: As you may or may not yet know, most experiments more or less fail and someone in the lab shouting “it worked!” is not so frequent. So it is kind of a miracle that your experiment worked quite ok. Do you have any ideas how to improve the workflow?
EG: First of all improve transformation efficiency – but we do not yet know at what step in the entire procedure. Yeast cells prefer the error-free pathway of homologous recombination. To get closer to the real question, the team was thinking to do the experiment in human cells where the choice between non-homologous end joining (NHEJ) and homologous recombination (HR) is not so biased. So that would definitely be more relevant to their question. But getting human cell cultures on the ISS would be another huge leap! We were just happy to get the yeast system working!
WN: You had mentioned before using organoids – which is definitely even more of a challenge.
EG: Well, after we have established yeast, the next step is clearly organoids! 😀
WN: Rebecca and Aarthi, you are now in university in biology or biomedical sciences. What are your future plans? Probably not going on a space mission?
RL: I’m going for Master’s degree in public health – that’s what I’ve been interested in for a long time – and then hopefully go to medical school and become a doctor.
AV: I am probably going into biophysics and biochemistry. I really like biochemistry research. Genes in Space has been the first time that I looked seriously into biochemistry and molecular biology and it definitely had a long term impact on me. I’d also like to go to med school and get an MD. I’d also like to stay in research and work on this kind of science for a while.
WN: Last question to Emily. What’s coming up in the next 500 applications for the Genes in Space contest?
EG: Actually my colleague Katy Martin is the Genes in Space program lead but it is still part of my job to support the program. We had more contests since this CRISPR-Cas project even though there were restrictions during the global pandemic. This year we will launch the experiment of the 2020 winner Kristoff Misquitta from New York. His experiment is based on the observation that pharmaceutical drugs may work less efficient in micro gravity and he proposed to investigate gene expression in the liver (where most drugs are metabolized) by quantitative PCR. Just recently, the 2021 winner of Genes in Space has been chosen. You can watch the presentations of the five finalists here: https://youtu.be/OWIXQ-sobKo.
WN: Thank you very much for answering all my questions and giving me further insights into your Genes in Space experiment. Good luck to Aarthi and Rebecca for their studies and their future career and for Emily, lots of interesting fun with the next generation of young Genes in Space scientists! It would be great if the European Space Agency would team up with Genes in Space so that we may also have German participants in the contest!
Genes in Space contest: genesinspace.org
Publication in PLOS One: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0253403
Press release – CRISPR proposal wins Genes in Space 2018 competition: https://www.genesinspace.org/news/press/genes-space-stem-competition-announces-winning-high-school-team/
Press release – Gene editing carried out on International Space Station: https://www.genesinspace.org/news/press/first-successful-crisprcas9-gene-editing-international-space-station/