BioScope

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Don’t Fear the CRISPR

Author: Sydney Wyatt

Editor: Keith Fraga

Human genetic modification strikes fear into many scientists and non-scientists alike. The recent claim of human genetic editing experiments suggested that a researcher in China, Dr. He Jiankui, edited human embryos to be resistant to HIV, and that some embryos were successfully carried to term (1). While the twin girls, and a potential third baby on the way from another couple, seem to be healthy, this may not be the first time Chinese researchers have genetically modified humans (2).

An RNA guide targets the Cas9 nuclease through complementary binding. The variable guide length allows for relatively easy design with high specificity to the target site. Genome Research Limited

Since the advent of CRISPR*, a specific genetic editing tool derived from a native bacteria defense system, genetic modification is the new hot ticket for research and media coverage (3). The technology is so accessible, citizen-scientists popularly known as biohackers have attempted to modify themselves with DIY CRISPR kits (4). While self-experimentation is discouraged, it begs the question: should scientists genetically modify a human using CRISPR?

Process of using recombinant DNA to engineer E. coli to produce human insulin for mass production. Genome Research Limited

Genetic editing is not a new technology. Recombinant DNA** has existed since the 1970s and is used extensively in research and biotechnology companies to create genetically modified organisms (5). For example, Genentech and Eli Lilly & Co used recombinant DNA to genetically modify bacteria in order to mass produce human insulin used to manage diabetes (approved by the FDA in 1982). Genetically modifying bacteria dramatically increased yield and purity of insulin over animal-sourced insulin, improving the access to this vital therapeutic for insulin patients.

At the time of its discovery, there was concern within the science community as well as within the general public over gene manipulation in humans using recombinant DNA. In February 1975, biologists, lawyers, and journalists gathered for the second Asilomar Conference on Recombinant DNA to draft regulations on experiments using recombinant DNA technology (6). Just prior to the conference, a moratorium on research projects using recombinant DNA had been voluntarily put in place and universally observed — a remarkable example of scientists’ ability to self-regulate (7). The recommendations that emerged from the conference addressed “how the scientific work could be undertaken with minimal risks to workers in laboratories, to the public at large, and to the animal and plant species sharing our ecosystems.”

Ultimately, the safety precautions were laid out in guidelines issued by the NIH in July 1976 but never became law despite legislators’ suggestions. Researchers instead continued self-regulation with the added guidance of the NIH Recombinant DNA Advisory Committee. It seemed like excessive red tape to deal with in order to perform any experiments using this technology, but the regulations were necessary to ensure public trust in scientific endeavors (7). In the decades since, these regulations have been refined to address new concerns in the field while maintaining forward momentum.

The far-reaching effects of the 1975 Asilomar Conference remains unmatched today. The new tech on the block is CRISPR, which is a far more nuanced and powerful genetic editing tool than recombinant DNA.

CRISPR/Cas9, commonly referred to as CRISPR in the media, is an engineered nuclease. And it’s not the first. Meganucleases (c. 1980s), zinc finger nucleases (ZFN; c. 1990s), and transcription activator-like effector nucleases (TALEN; c. 2010) make up the larger family of engineered nucleases. ZFNs, TALENs, and CRISPR (c. 2013) have been used successfully for genetic editing (8).

Zinc fingers (ZF) recognize codons, or 3-base codes in the DNA, to guide the FokI nuclease to the cut site. Genome Research Limited

ZFNs were the first truly programmable genetic editing tool and was applied in humans to disrupt the CCR5 co-receptor that HIV uses to enter cells. Preclinical trials using ex vivo somatic cell gene editing successfully demonstrated that using ZFN gene therapy for HIV treatment was feasible and safe and thus merited clinical trials with the intent to take this treatment to market (9). Phase I and Phase II clinical trials are being conducted in the United States regarding the treatment of HIV patients; clinical trials are also underway for several other diseases (10). Importantly, these trials edit only somatic cells of the patient, not the germline cells (eggs or sperm), so the changes made will not be inherited. I spoke with gene therapy researcher Dr. David Segal of University of California, Davis, about these clinical trials.

“The things that go into clinical trial, by government regulation, have to pass certain thresholds for safety and efficacy…We strive to say anything that goes into a person is as safe as we know it to be. So everyone should know [that] about clinical trials. They go to great lengths to demonstrate safety above all else – even before efficacy – when you go into clinical trial.” – Dr. David Segal

TLN sites recognize single bases, allowing for more freedom in choosing targets for editing with the FokI nuclease. Genome Research Limited

TALENs were next on the scene, but were quickly outshined by CRISPR. Thus, human genetic editing using TALENs has not made it to clinical trials in the United States.

This brings us full circle to CRISPR’d humans. It is interesting that He Jiankui, the man behind the CRISPR babies, also chose to target CCR5. Since the original MIT Technology Review article was published, a January 21 report from Xinhua, China’s state media agency, has confirmed the birth of genetically edited twin girls and the pregnancy of another couple, suggesting a third genetically edited could be born (11).

Was this ethical? According to the report, no. Segal elaborated on the ethical measures we have regarding genetic editing in humans and that He bypassed these ethical measures for his germline editing experiments:

“[W]hen science starts to push up against ethical boundaries, we have institutions in place to try to respect, society’s concerns about the research that are being done…it’s absolutely essential that all the work go through this committee…anything you do that involves people, even if it’s a psychological questionnaire that at the worst case could cost someone some mental harm, all the way up to working with human embryos…has to be approved by an institutional review board. And again, they want to maintain transparency…They are the oversight at this institution. They have different people on the committee. There’s kinds of protections that they look for. All the people doing the research need to have training in ethics of human research that involves the unethical behaviors that had been done in the past and how we need to avoid that. If we don’t train the investigators, if we don’t go through the IRB, if we don’t follow this procedures, these institutions that have been set up to maintain transparency and trust and the scientific endeavor in the eyes of society, the investigator could lose his job. The NIH can stop funding the entire university. I mean, there are big consequences [in] trying to circumvent these structures that have been put in place to maintain society’s confidence in the scientific ventures. And I would say that was the most egregious thing that scientist in China did.”

He Jiankui blatantly broke these agreements, resulting in the ethical uproar over his experiments. Segal also discussed the long-term consequences of germline gene editing and gene therapy in our interview, which can be read in full here.

Interestingly, a group of scientists, including a few of the original discoverers of CRISPR and recombinant DNA, issued a statement published in Science in 2015 requesting a moratorium on human genetic modification (12). Echos of the recommendations from the Asilomar Conference are present in the recommendations laid out in the statement. According to Segal, some of these recommendations have come to pass – CRISPR research transparency is highly encouraged through the establishment of forums like the International Summit on Human Genome Editing where He unveiled his work.

The recommendations: 1) Discourage, even in countries that might be permissive, any research aimed at heritable genetic modification; 2) Establish forums for information and education on the risks and rewards of using CRISPR to treat or cure human disease, and the accompanying ethical, social, and legal concerns; 3) Support research transparency to help determine whether or which clinical applications are permissible; 4) Gather experts in genetics, law, and bioethics as well as members of the scientific community and public at large to consider the issues at hand.

A publication from 2017 discussed similar concerns about whether we are prepared for CRISPR clinical trials (13). The publication mentions a Phase I clinical trial was already in progress in China with the intention of treating stage IV metastatic non-small cell lung cancer, but that a similar trial was still prospective in the United States. The authors offered an extensive and critical review of the preclinical data that was presented to the NIH Recombinant DNA Advisory Committee (sound familiar? It’s the same committee that was established around the time of the Asilomar Conference).

In short, they determined we are not ready. This conclusion was informed by an existing framework for assessing the jump from preclinical to clinical trials (14-17).

Yet on February 1, NPR published an article on human genetic editing that is happening in the United States (18). Dieter Egli of Columbia University claims that he is conducting his experiments genuinely for research, in contrast to He’s supposed goal of genetically protecting the babies from HIV. Currently, Egli is focused on correcting one of the underlying genetic defects that result in inherited blindness and only allows the modified embryos to develop for one day, though he hopes to allow further development if these initial experiments are successful.

Federal funds are banned from being used for this kind of research – germline editing – in the United States, but there is no such stipulation on private funding (i.e. self-funded, as implied in the case of He’s work). If you recall, DIY CRISPR kits are readily available online, so if one has the means, then there is nothing preventing DIY designer babies beyond one’s ethics. There is still considerable controversy: should the moratorium be instilled? Should it be applied to basic research, such as Egli’s project? Can adequate regulations be established to prevent the need for a moratorium?

According to Segal, “we’re in the very early days of trying to use this as a therapy. Most of the work doesn’t involve any humans, but some things are progressing to a point where it can be used in humans, and in clinical trial.”

Only time will tell. In the meantime, don’t fear the CRISPR. It won’t be coming to a human near you anytime soon.

Thank you Dr. Segal for taking the time to provide his expert opinion on this topic.

 

*CRISPR: a genetic engineering tool using a short, repetitive DNA sequence and associated editing protein Cas9 to specifically edit target DNA sequence.

**Recombinant DNA: DNA made by artificially combining DNA fragments from different organisms.

 

For more history on genetics, check out The Gene by Siddhartha Mukherjee.

Bibliography:

 

  1. Regalado, A. (2018, November 26). EXCLUSIVE: Chinese scientists are creating CRISPR babies.
  2. Foley, K. E. (2018, January 26). Chinese scientists used Crispr gene editing on 86 human patients.
  3. Doudna, J. (2015, January). Genome Engineering with CRISPR-Cas9: Birth of a Breakthrough Technology.
  4. Lee, S. M. (2019, January 17). This Biohacker Is Trying To Edit His Own DNA And Wants You To Join Him.
  5. Herbert W. Boyer and Stanley N. Cohen. (2017, December 11).
  6. Berg, P., Baltimore, D., Brenner, S., Roblin, R. O., III, & Singer, M. F. (1975). Summary Statement of the Asilomar Conference on Recombinant DNA Molecules. PNAS, 72(6), 1981-1984. doi:10.1073/pnas.72.6.1981
  7. The Paul Berg Papers: Recombinant DNA Technologies and Researchers’ Responsibilities, 1973-1980. (n.d.).
  8. Chandrasegaran, S., & Carroll, D. (2016). Origins of Programmable Nucleases for Genome Engineering. Journal of Molecular Biology, 428(5), 963-989. doi:10.1016/j.jmb.2015.10.014
  9. DiGiusto, D. L., Cannon, P. M., Holmes, M. C., Li, L., Rao, A., Wang, J., . . . Zaia, J. A. (2016). Preclinical development and qualification of ZFN-mediated CCR5 disruption in human hematopoietic stem/progenitor cells. Molecular Therapy — Methods & Clinical Development, 3. doi:10.1038/mtm.2016.67
  10. Search of: Zfn | United States – List Results. (n.d.).
  11. Cross, R. (2019, January 21). Rogue CRISPR scientist will be punished. C&EN97(3), 1-56.
  12. David, B., Berg, P., Botchan, M., Carroll, D., Charo, R. A., Church, G., . . . Yamamoto, K. R. (2015). A prudent path forward for genomic engineering and germline gene modification. Science,348(6230), 36-38. doi:10.1126/science.aab1028
  13. Baylis, F., & McLeod, M. (2017). First-in-human Phase 1 CRISPR Gene Editing Cancer Trials: Are We Ready? Current Gene Therapy, 17, 309-319. doi:10.2174/1566523217666171121165935
  14. Kimmelman J. (2009). Gene transfer and the ethics of first-in-human research: lost in translation. Cambridge University Press.
  15. Henderson V.C., Kimmelman J., Fergusson D., Grimshaw J.M., & Hackam D.G. (2013). Threats to validity in the design and conduct of preclinical efficacy studies: A systematic review of guidelines for in vivo animal experiments. PLoS Med, 10(7):e1001489.
  16. Kimmelman J., & Henderson V. (2015). Assessing risk/benefit for trials using preclinical evidence: a proposal. J. Med. Ethics, 42(1), 50.
  17. Kimmelman J., & London A.J. (2011). Predicting harms and benefits in translational trials: ethics, evidence, and uncertainty. PLoS Med, 8(3):e1001010.
  18. Stein, R. (2019, February 01). New U.S. Experiments Aim To Create Gene-Edited Human Embryos. Retrieved from https://www.npr.org/sections/health-shots/2019/02/01/689623550/new-u-s-experiments-aim-to-create-gene-edited-human-embryos

Finding Your Way: Choosing a Thesis Lab

Contributing authors (alphabetical order): Emily Cartwright, Anna Feitzinger, Keith Fraga, Hongyan Hao, Jessica Huang, Sharon Lee, Linda Ma

 

Congratulations, you’ve made it past the harrowing applications, nerve-wracking interviews, and awesome recruitment food! The first year in graduate school can be difficult, as you juggle coursework with organizing rotations and looking for the lab you’ll be dedicating the next 4-6 years of your life to. The question of which lab you will join is the question that you think about all the time, and rightfully so. The experience and relationships you make during your PhD are transformative. But there are many variables to consider and reaching a final decision on which lab to join can be a challenge!

All of us here at BioScope have gone through the same process, and we have some ideas that just might give first-year graduate students another perspective on deciding on a lab. In some ways, this is an advice column, but definitely not a “How to” article. We don’t know of a magic bullet that makes this decision easy. Part of the process is actually experiencing the process itself: all of the highs and lows, all of the epiphanies and backtracking, and the feeling of finally deciding. So let’s get started!

Getting through rotations!

Perhaps the first step in deciding on a lab is doing rotations. Granted, some disciplines and graduate programs do not operate on a formal rotation schedule. However, in general, there is a period of time during the first year where you will have the chance to rotate with a lab that you are interested in. The “Car Dealership Test Drive” analogy works perfectly here. What better way to experience a lab environment, the research they do, and how you work with the PI more than doing a rotation with them?

Specifically, here at UC Davis, many graduate programs provide great resources for finding faculty to rotate with. Don’t forget that you can look in other departments as well! UC Davis has diverse faculty, covering a range of fields that you will definitely find something that you’re interested in pursuing.

While shopping for a car, you can test drive as many cars as you want, which is not the case in graduate school. You can only participate in a limited number of rotations. Therefore, there can be a lot of planning and reflection that goes into who you should rotate with. There are two things we want to stress about managing rotations:

(1) Know what you want to study – or what you DON’T want to study – to a point where you can narrow down the labs you are interested in. Having the self-discipline to focus your interests is critical for decisive rotation decisions.

(2) Rotations are less about the progress you make in the short amount of time in the lab, and more about getting a feel for the lab. In the rotation, you receive a small project, and in the hopes of impressing the PI, the lab, and your peers, you devote a lot of energy to generating results. Striking gold during a rotation (such as getting results that will contribute to a manuscript) is rare, and not something to bet on. Instead, it is much more efficient to devote your attention to being in lab, experiencing the group, learning how you work with the PI, and gaining a solid grasp of the research program.

Honesty and Realism goes a long way

An important thing to remember when looking for rotation labs is to be realistic. You can’t be searching for a lab that studies microRNAs in brain development by day and cures Down syndrome by night. Even a lab with diverse projects maintains very specific and well-defined areas of interest. Having ambitious goals and ideas are great, but the key is to not pigeon-hole yourself to a point where every lab you come across doesn’t quite do everything you are excited about. Your perfect lab does not exist. You have to let your scientific interests grow and develop, and allow yourself to be mentored.

This goes to a deeper point about graduate school. It is not so much what you do, but how you do it and learning the skills to be a scientist. It is very common for individuals to work on something totally different from their thesis research after graduating. Doing a PhD helps you sharpen the tools to study a variety of problems. Getting a PhD is more about the training, and finding a lab is more about fit than it is about field.

Handling uncertainty is key

As mentioned above about rotations, knowing your interests is key to to deciding which lab to join. This is part of “Knowing yourself,” which consists of answers to questions like, “What am I interested in?”, “What type of research environment do I work best in?”, “Do I want a PI that is hands-on or hands-off?”, “What do I want to do after my PhD? And how can my thesis lab facilitate that?”, and many more. You can answer these questions by thinking back to labs you’ve worked in before and what you liked and didn’t like about them. With each rotation, you’ll be able to get a clearer idea of what your answers to these questions are.

Deciding on a thesis lab can be roughly split into concerns of two types: concerns that you can control, and concerns that are beyond your control. What we want to highlight is an appreciation of the difference between what you have control over (your attitudes, your interests, your effort) and things you cannot control or predict (how your relationship with your PI will develop, how funding will change, how experiments will go).

The major pitfall in deciding a thesis lab is being too worried about things that you cannot control. We are all concerned about choosing the “wrong” lab, becoming stuck in a situation where we need to switch advisors. In those cases, the relationship with the PI deteriorates due to a host of reasons. You cannot forecast these changes to your relationships. You do your best to address problems early on and find solutions. Appreciating the things that you can control gives you a tool in making your decision.

Things you should ask yourself

Choosing a thesis lab is a very personal process. It is about you, and you finding your way through graduate school. All of us here at BioScope arrived at our respective labs in different ways, and we pondered different concerns. However, we have recognized a few questions you should ask yourself. These are questions that you do not need the answer to right now. These are questions where your answer will change over time, maybe every 5 minutes! Nevertheless, these are some questions that are meant to get you thinking.

The Research

  • Do I love the science, and am I excited about the unanswered questions in the field?
  • Can I see myself truly enjoying reading papers in this field?
  • Am I willing to perform the literature searches necessary to fill in my gaps of knowledge?
  • Can I imagine performing the essential lab techniques on a daily basis, becoming an expert in the lab’s tools?

The Thesis Advisor

  • Can I see myself working with this PI for the next several years?
  • How comfortable do I feel communicating with the P.I.? Is it easy to have a conversation and brainstorm ideas?
  • Do I do better when the P.I. has an open door policy (questions always welcome), or can I be productive without meeting with my P.I. once a week?
  • What is the PI’s track record with other PhD students? What do other people say about the lab?
  • Is my PI supportive of my future goals?

The Environment

  • Can I have enjoyable and intellectual communications with the other students/post-docs, or do I feel like there are unpleasant interactions?
  • Do I prefer a lab that’s more social, or one where everyone goes into lab just to get the work done?

The grass on the other side is still just grass

Finally settling into a lab is a wonderful feeling; It’s like finally finding a home. And yet, we still have our difficulties. We still have our miscommunication. We still sometimes ponder if we made the right decision. It is very natural to have these questions because, like we said, there is no magic bullet, no recipe for doing this. And as scientists that strive for some degree of precision and exactness in our lives, this is hard to wrestle with. A lab is not perfect when you join. It takes dedication, patience, and communication to create a PhD training perfect for you. So keep calm, and carry on.

 

 

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