Author: srwyatt

Don’t Fear the CRISPR

On March 14, 2019

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:

Regalado, A. (2018, November 26). EXCLUSIVE: Chinese scientists are creating CRISPR babies.
Foley, K. E. (2018, January 26). Chinese scientists used Crispr gene editing on 86 human patients.
Doudna, J. (2015, January). Genome Engineering with CRISPR-Cas9: Birth of a Breakthrough Technology.
Lee, S. M. (2019, January 17). This Biohacker Is Trying To Edit His Own DNA And Wants You To Join Him.
Herbert W. Boyer and Stanley N. Cohen. (2017, December 11).
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
The Paul Berg Papers: Recombinant DNA Technologies and Researchers’ Responsibilities, 1973-1980. (n.d.).
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
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
Search of: Zfn | United States – List Results. (n.d.).
Cross, R. (2019, January 21). Rogue CRISPR scientist will be punished. C&EN, 97(3), 1-56.
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
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
Kimmelman J. (2009). Gene transfer and the ethics of first-in-human research: lost in translation. Cambridge University Press.
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.
Kimmelman J., & Henderson V. (2015). Assessing risk/benefit for trials using preclinical evidence: a proposal. J. Med. Ethics, 42(1), 50.
Kimmelman J., & London A.J. (2011). Predicting harms and benefits in translational trials: ethics, evidence, and uncertainty. PLoS Med, 8(3):e1001010.
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

Fruit Flies’ All Time Favorite Fruit

By srwyatt

On February 12, 2019

In main_pub

Author: Anna Feitzinger

Editor: Sharon Lee

Undeniably, you have seen a flurry of little fruit flies whizzing around left out bananas or oranges on a kitchen table. This little pest has been co-living, or commensal, with humans for thousands of years. But almost all of Drosophila melanogaster’s closest cousins, with the exception of Drosophila simulans, don’t enjoy human company (1) – so how did this specific species become so chummy with man? Researchers from Lund University have published their discoveries (2) of previously unknown aspects of the natural ecology of one of the longest standing model organisms, which may explain how D. melanogaster became a human commensal and spread across the world.

Like humans, D. melanogaster has its origins in Africa. D. melanogaster migrated out of Africa and began to colonize Europe and Asia an estimated 10,000 years ago (3) . Given the commensal nature of D. melanogaster, it is no surprise that it has become one of the most widely used model organisms. Despite it’s annoyance around ripened fruit, the chromosomal theory of inheritance, genetic control of early development, and fundamental principles in neurobiology, population and evolutionary genetics all have a basis in D. melanogaster research. Given the century-long use of D. melanogaster in the laboratory, it is surprising that few fundamental aspects of the natural ecology of D. melanogaster is known. Interactions of organisms with their environment drive adaptation, shaping their evolution and inevitably the present day biology that we study. It has been said that D. melanogaster lives where it eats, and for the first time we now know the likely ancestral host of D. melanogaster: the marula fruit.

Suzan Mansourian and colleagues set out to Matopos National Park in Zimbabwe to determine the host fruit of wild South African populations of D. melanogaster. The extremely abundant marula fruit has a similar pH to an orange, a highly fermentable pulp and contains terpenes and esters which are known olfactory cues for D. melanogaster. These characteristics, paired with the fact that domesticated D. melanogaster’s favorite breeding substrate is the orange, made the marula fruit the perfect candidate. Indeed, wild D. melanogaster were found in fly traps containing marula placed on the forest floor.

To test the hypothesis that flies from native habitats prefer marula to other fruit, the researchers placed paired traps containing either marula or orange under fruiting marula trees. D. melanogaster showed a strong preference for the marula fruit, although their sister species D. simulans, also native to the region, did not. Traps containing marula placed in locations with no fruiting marula trees, but other fruiting trees, caught none or very few D. melanogaster. These experiments have garnered D. melanogaster a new title: seasonal specialists!

How do domesticated flies react to marula fruit? Canton-S strain flies, originally collected sometime before 1916 in Canton, Ohio, prefer marula when given the choice between orange and marula. This conserved preference is impressive given that marula is not found outside sub-saharan Africa. To determine what specific chemical mediates this preference, the researchers tested major chemical components in a two-choice assay and found a high preference of esters responsible. Furthermore, using functional imaging of transgenic flies, the primary marula ester ethyl isovalerate was shown to activate Or22a-expressing olfactory sensory neurons (ab3a/b). In contrast, odor from oranges triggered weak to no activity. Silencing of the Or22a pathway using RNAi reduced the ability of flies to localize to marula compared to controls, suggesting that this pathway is responsible for detection and localization to marula.

If the Or22a circuit is linked to the specific chemistry of host fruit, Mansourian et al reasoned that local adaptation of the Or22a receptor would be found in fly populations from environments which contain different hosts. To investigate this, olfactory receptors sequences from 10 different African genomes and 1 European genome were analyzed. They found that Or22a and its adjacent paralog Or22b indeed showed genetic differentiation, a sign of local adaptation, between populations in contrast to other olfactory receptors. Next, they wanted to know if the sequence differences in Or22a and Or22b between populations confer functional changes. Measurements from ab3a neurons in a strain carrying a prevalent African variant of the receptor, Or22a/b (a fused gene as a result of a deletion), was found to be even more sensitive to the marula ester than non-African flies, confirming that the genetic differences are functional.

The now-vanished San tribes, known for their elaborate cave paintings, inhabited the Matopos during the Late Pleistocene to Early Holocene periods. Like D. melanogaster, the San appear to also have been seasonal specialists on marula – in one cave alone at least 24 million marula pits were recovered. This link may explain how D. melanogaster became a human commensal – wandering into San inhabited caves to feast on marula. Interestingly, traps placed inside the caves caught a number of D. melanogaster, but not D. simulans. The estimated date of the within-Africa expansion of D. melanogaster corresponds roughly with the date that marula harvesting ceased. D. melanogaster may have become dependent on marula harvesting by the San and when this ceased left the region with the San as a human commensal. Thus, the marula fruit may have been the glue that brought humans and one our most beloved model organisms into cohabitation, sparking their migration across the world and eventually into our present day laboratories.

Descendants of the ancient San have long been an interest to researchers. A 2010 paper in Nature showed that there is more genetic diversity between two San genomes than between an Asian and European genome (4). More recently, the San people became the first African group to draft a code of ethics for researchers who use their sequencing data (5). Although the use of the marula fruit by the ancient San ceased about ~10,000 years ago, it is still used commercially today both in beverages and cosmetics. Perhaps enjoying amarula, a cream liqueur made from marula, will help give a taste of what brought man and D. melanogaster together.

Author: Anna Feitzinger

Keller A (2007) Drosophila melanogaster’s history as a human commensal. Curr Biol 17(3):77–81.
Mansourian S, et al. (2018) Wild African Drosophila melanogaster Are Seasonal Specialists on Marula Fruit. Curr Biol 28(24):3960–3968.e3.
Pool JE, et al. (2012) Population Genomics of Sub-Saharan Drosophila melanogaster: African Diversity and Non-African Admixture. PLoS Genet 8(12). doi:10.1371/journal.pgen.1003080.
NordlingMar L (2017) San people of Africa draft code of ethics for researchers. Science | AAAS. Available at: http://www.sciencemag.org/news/2017/03/san-people-africa-draft-code-ethics-researchers [Accessed February 6, 2019].
Schuster SC, et al. (2010) Complete Khoisan and Bantu genomes from southern Africa. Nature 463(7283):943–947.

San Code of Ethics available to read here: http://www.globalcodeofconduct.org/affiliated-codes/

A Day of Biology and Mathematics in the Bay Area

By srwyatt

On December 17, 2018

In main_pub

Saturday, November 3rd marked the 12th Annual Biology and Mathematics in the Bay Area Conference (BaMBA). BaMBA has developed into a particularly unique conference where you can expect high-level research and opportunities for interdisciplinary collaboration. BaMBA 2018 was held at the incredible Clark Center at Stanford University.

This year’s BaMBA meeting featured five fantastic experts presenting topics ranging from statistical methods in machine learning to cell biology of the immune system: Dr. Lacramioara Bintu (Stanford University) Dr. Sean Collins (UC Davis), Dr. Bin Yu (UC Berkeley), Dr. Dexter Hadley (UC San Francisco), and Dr. Andrew Fire (Stanford University).

I had a chance to speak with Dr. Massa Shoura of Stanford University, an organizer for BaMBA 2018, about what makes BaMBA unique. “What I like about it (sic) brings people together that usually don’t go to the same conferences.” Interdisciplinary conferences like BaMBA are important opportunities for attendees to gain new perspectives on their research.

“As we grow older scientifically we end up specializing and then we end up going to the same conferences and talking to the same people because we are trying to solve something very intricate, but it is nice every now and then for us to get out of our comfort zone and talk to people who we usually dont talk and we find out this person can look at the same problem from a different angle and we end up having diversity in the way we solve the problem.” – Dr. Shoura

Dr. Shoura also pointed out that, because BaMBA is funded from several sources such as UC Davis College of Biological Sciences, the registration fee is waived for all attendees, allowing a broad audience of students, professors, and professionals from across the Bay Area to attend.

Diana Sernas, a 4th year undergraduate student at UCSC (‘19), also thought the accessibility of BaMBA was an important element to the conference’s uniqueness and success. According to her, “[a]ny opportunity for undergraduates to present their work is just another [opportunity for] professional development.” She also presented a research poster about her work on analyzing the 3D architecture of the genome with Dr. Javier Arsuaga.

“Presenting a poster at a conference is a great way for undergraduates to meet other people passionate about science and to see where they might fit in. “We are having an opportunity to look like a scientist, to be a scientist, to present ourselves as scientists.” – Diana Sernas (‘19)

I was especially excited for Dr. Sean Collins’ presentation from the UC Davis Department of Molecular & Cellular Biology. Dr. Collins’ research focuses on understanding the complex regulation of neutrophil chemotaxis vital in the immune response. With the use of live-cell reporters, the Collins lab is able to quantitatively interrogate how neutrophils locate pathogens in complex, noisy environments.

Dr. Collins also pointed out the significant impact BaMBA’s accessibility and well-organized schedule has had on its success: “I think it’s a really great meeting. It’s a nice scale [and] easy to interact with people [even] with a full day… I have also been enjoying breadth of the meeting with really different talks and posters. It’s very nice.”

In addition to the main speakers, sessions/panels, and poster sessions, Q&A sessions offered time to discuss the material and, in some cases, resulted in initiating collaborative projects. Dr. Bin Yu of UC Berkeley “really enjoyed [BaMBA]” and was especially happy “to talk to students and [make] professional connections with other speakers.”

I highly recommend attending BaMBA in the future! It is an unique, accessible conference and an incredible opportunity to meet other passionate scientists. Keep an eye out for information about future BaMBA conferences at the the website: bambameeting.org. You can expect to see me there at BaMBA 13!

Author: Keith Fraga

Editor: Sydney Wyatt

A Video is Worth 1,000 Pictures

By srwyatt

On October 29, 2018

In main_pub

Still of HIV virus from The HIV Life Cycle.

Dr. Janet Iwasa from University of Utah gave a stellar keynote address on animating biology for the annual Molecular & Cellular Biology Training Program retreat on Friday, October 19. The major challenge for science communicators is to explain complex abstract concepts in a non-specialist manner. Whether it’s explaining the origin of life or how HIV infects the body, animation will revolutionize science communication, according to Dr. Iwasa. This form of visualization makes material tangible to a non-specialist audience and truly captivates their attention. Many students at the retreat were inspired to start our animation careers immediately, including myself. However, Dr. Iwasa offered sage advice — “Build yourself a comfortable little niche…Think about what skills you have now, and how those could be applied to a new discipline or area of research to create a unique niche.” Though this curbed my short-lived dream of becoming a world-famous biology animator, her material is freely available to download.

Author – Sydney Wyatt

Editor – Keith Fraga