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CRISPR Conundrum: Pursuing Consensus on Human Germline Editing

June 12, 2020

By Daniel Erenstein, Neurobiology, Physiology, and Behavior, ‘21

Author’s Note: In November 2018, a scientist in China became the first person to claim that they had edited the genes of human embryos carried to term. Two twins, named with the pseudonyms Lulu and Nana, were born from these very controversial experiments. This news rapidly propelled the debate on human germline genome editing into the mainstream. My interest in this issue was inspired by my involvement with the Innovative Genomics Institute, located in Berkeley, CA. While attending Berkeley City College during the spring and fall semesters of 2019, I participated in the institute’s CRISPR journal club for undergraduates. Each week, we discussed the latest research from the field of CRISPR gene editing. I also took part in a conference, attended by leading geneticists, bioethicists, philosophers, professors of law and policy, science journalists, and other stakeholders, examining where the consensus, if any, lies on human germline genome editing. Discussions from this conference serve as a foundation for this submission to The Aggie Transcript.

 

New details have emerged in the ongoing controversy that kicked off in November of 2018 when a Chinese biophysicist claimed that, during in vitro fertilization, he had genetically edited two embryos that were later implanted into their mother. Twins, anonymously named Lulu and Nana, are believed to have been born as a result of these experiments. This announcement from He Jiankui in a presentation at the Second International Summit on Human Germline Editing in Hong Kong was largely met with swift condemnation from scientists and bioethicists [1, 2].

Late last year, excerpts of the unpublished research report were made public for the first time since He’s announcement, shedding light on his approach to edit resistance to human immunodeficiency virus, or HIV, into human genomes using CRISPR-Cas9 [3]. CRISPR, short for clustered regularly interspaced short palindromic repeats, are specific patterns in bacterial DNA. Normally, a bacterium that has survived an attack by a bacteriophage—a virus that infects bacteria and depends on them in order to reproduce—will catalog bacteriophage DNA by incorporating these viral sequences into their own DNA library. This genetic archive of viral DNA, stored between the palindromic repeats of CRISPR, can be revisited as a reference when the bacterium faces future attacks, aiding in its immune response [4].

To respond effectively, bacteria will transcribe a complementary CRISPR RNA molecule from the existing CRISPR sequence. Using crRNA—short for CRISPR RNA—as a guide, CRISPR-associated proteins play the part of a search engine, scanning the cell for any entering viral DNA that matches the crRNA sequence [5]. There are many subtypes of CRISPR-associated proteins [6], but Cas9 is one such type that acts as an enzyme by catalyzing double-stranded breaks in sequences complementary to the guide [7]. This immune system effectively defends against the DNA of invading bacteriophages, protecting the bacterium from succumbing to the virus [5]. 

A cell’s built-in mechanisms typically repair any double-stranded breaks in DNA via one of two processes: nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) [8]. During NHEJ, base pairs might be unintentionally inserted or deleted, causing frameshift mutations called indels in the repaired DNA sequence. These mutations significantly affect the structure and function of any protein encoded by the sequence and can result in a completely nonfunctional gene product. NHEJ is frequently relied upon by gene editing researchers to “knock out” or inactivate certain genes. HDR is less efficient, but the process is often exploited by scientists to “knock in” genes or substitute DNA [9].

CRISPR is programmable, meaning that certain DNA sequences can be easily added to these sites, precisely altering the cell’s genetic code at specific locations. Jiankui He was not the first to use CRISPR to edit the genes of human embryos, but no one was known to have ever performed these experiments on viable embryos intended for a pregnancy. He and two of his colleagues have since been fined and sentenced to prison for falsifying ethical review documents and misinforming doctors, the state-run Chinese news agency Xinhua reported in December 2019 [10]. But He’s experiments supposedly yielded another birth during the second half of 2019 [11], confirmed by China in January [12], and Russian scientist Denis Rebrikov has since expressed strong interest in moving forward with human germline genome editing to explore a potential cure for deafness [13].

Despite what seems like overwhelming opposition to human germline genome editing, He’s work has even generated interest from self-described biohackers like Josiah Zayner, CEO of The ODIN, a company which produces do-it-yourself genetic engineering kits for use at home and in the classroom. 

“As long as the children He Jiankui engineered haven’t been harmed by the experiment, he is just a scientist who forged some documents to convince medical doctors to implant gene-edited embryos,” said Zayner in a STAT opinion reacting to news of He’s sentence [14]. “The 4-minute mile of human genetic engineering has been broken. It will happen again.”

Concerns abound, though, about the use of this technology to cure human diseases. And against the chilling backdrop of a global COVID-19 pandemic, fears run especially high about bad actors using CRISPR gene editing with malicious intent. 

“A scientist or biohacker with basic lab know-how could conceivably buy DNA sequences and, using CRISPR, edit them to make an even more panic-inducing bacteria or virus,” said Neal Bear, a television producer and global health lecturer at Harvard Medical School, in a recent STAT opinion [15]. “What’s to stop a rogue scientist from using CRISPR to conjure up an even deadlier version of Ebola or a more transmissible SARS?” 

Into the unknown: understanding off-target effects

In his initial presentation, He said that he had targeted the C-C chemokine receptor type 5 (CCR5) gene, which codes for a receptor on white blood cells recognized by HIV during infection. His presentation suggested that gene editing introduced a known mutation named CCR5Δ32 that changes the receptor enough to at least partially inhibit recognition by HIV. The babies’ father was a carrier of HIV, so this editing was performed to supposedly protect the twins from future HIV infection [16]. 

He’s edits to the CCR5 gene—and human germline genome editing, in general—worry geneticists because the off-target effects of introducing artificial changes into the human gene pool are largely unknown. In a video posted on his lab’s YouTube channel [17], He claimed that follow-up sequencing of the twins’ genomes confirmed that “no gene was changed except the one to prevent HIV infection.”

Excerpts from the unpublished study indicate otherwise, according to an expert asked to comment on He’s research in MIT Technology Review, because any cells taken from the twins to run these sequencing tests were no longer part of the developing embryos [3].

“It is technically impossible to determine whether an edited embryo ‘did not show any off-target mutations’ without destroying that embryo by inspecting every one of its cells,” said Fyodor Urnov, professor of molecular and cell biology at UC Berkeley and gene-editing specialist [3]. “This is a key problem for the entirety of the embryo-editing field, one that the authors sweep under the rug here.”

Urnov’s comments raise concerns about “mosaicism” in the cells of Lulu and Nana—and any other future babies brought to term after germline genome editing during embryonic stages of development. In his experiments, He used preimplantation genetic diagnosis to verify gene editing. Even if the cells tested through this technique showed the intended mutation, though, there is a significant risk that the remaining cells in the embryo were left unedited or that unknown mutations with unforeseeable consequences were introduced [16].

While the CCR5Δ32 mutation has, indeed, been found to be associated with HIV resistance [18, 19], even individuals with both copies of CCR5Δ32 can still be infected with certain strains of HIV [20]. In addition, the CCR5Δ32 mutation is found almost exclusively in certain European populations and in very low frequencies elsewhere, including China [21, 22], amplifying the uncertain risk of introducing this particular mutation into Chinese individuals and the broader Chinese gene pool [16].

Perhaps most shocking to the scientific community is the revelation that He’s experiment did not actually edit the CCR5 gene as intended. In He’s November 2018 presentation, he discussed the rates of mutation via non-homologous end-joining but made no mention of the other repair mechanism, homology-directed repair, which would be used to “knock in” the intended mutation. This “[suggests] that He had no intention of generating the CCR5Δ32 allele,” wrote Haoyi Wang and Hui Yang in a PLoS Biology paper on He’s experiments [16].

Gauging the necessity of germline genome editing

The potential of CRISPR to revolutionize how we treat diseases like cystic fibrosis, sickle cell disease, and muscular dystrophy is frequently discussed in the news; just recently, clinical trials involving a gene-editing treatment for Leber congenital amaurosis, a rare genetic eye disorder, stirred enthusiasm, becoming the first treatment to directly edit DNA while it’s still in the body [23]. While this treatment edits somatic cells—cells that are not passed onto future generations during reproduction—there is increasing demand for the use of germline genome editing as well, even despite the reservations of scientists and bioethicists. 

This begs the question: how will society decide what types of genetic modifications are needed? In the case of He’s experiments, most agree that germline genome editing was an unnecessary strategy to protect against HIV. Assisted reproductive technology (ART), a technique that features washing the father’s sperm of excess seminal fluids before in vitro fertilization (IVF), was used in He’s experiments [3] and has already been established as an effective defense against HIV transmission [24]. Appropriately handling gametes—another word for sperm and egg cells—during IVF is an additional method used to protect the embryo from viral transmission, according to Jeanne O’Brien, a reproductive endocrinologist at the Shady Grove Fertility Center [3].

“As for considering future immunity to HIV infection, simply avoiding potential risk of HIV exposure suffices for most people,” wrote Wang and Yang in their PLoS Biology paper [16]. “Therefore, editing early embryos does not provide benefits for the babies, while posing potentially serious risks on multiple fronts.”

One such unintended risk of He’s experiments might be increased susceptibility to West Nile virus, an infection thought to be prevented by unmutated copies of the CCR5 receptor [11]. 

In a paper that examines the societal and ethical impacts of human germline genome editing, published last year in The CRISPR Journal [25], authors Jodi Halpern, Sharon O’Hara, Kevin Doxzen, Lea Witkowsky, and Aleksa Owen add that “this mutation may increase vulnerability to other infections such as influenza, creating an undue burden on these offspring, [so] we would opt instead for safer ways to prevent HIV infection.”

The authors go on to propose the implementation of a Human Rights Impact Assessment. This assessment would evaluate germline editing treatments or policies using questions that weigh the benefits of an intervention against its possible risks or its potential to generate discrimination. The ultimate goal of such an assessment would be to “establish robust regulatory frameworks necessary for the global protection of human rights” [25].

Most acknowledge that there are several questions to answer before human germline genome editing should proceed: Should we do it? Which applications of the technology are ethical? How can we govern human germline genome editing? Who has the privilege of making these decisions?

Evaluating consensus on germline genome editing

In late October of last year, scientists, bioethicists, policymakers, patient advocates, and religious leaders gathered with members of the public in Berkeley for a discussion centered around some of these unanswered questions. One of the pioneers of CRISPR gene editing technologies, Jennifer Doudna, is a professor of biochemistry and molecular biology at UC Berkeley, and the Innovative Genomics Institute, which houses Doudna’s lab, organized this CRISPR Consensus? conference in collaboration with the Initiative on Science, Technology, and Human Identity at Arizona State University and the Keystone Policy Center. 

The goal of the conference was to generate conversation about where the consensus, if any, lies on human germline genome editing. One of the conference organizers, J. Benjamin Hurlbut, emphasized the role that bioethics—the study of ethical, social, and legal issues caused by biomedical technologies—should play in considerations of germline genome editing. 

He’s “aim was apparently to race ahead of his scientific competitors but also to reshape and speed up, as he put it, the ethical debate. But speed is surely not what we need in this case,” said Hurlbut, associate professor of biology and society at Arizona State University, at the conference [26].

Central to the debate surrounding consensus is the issue of stakeholders in decision-making about germline genome editing. Experts seem to be divided in their definitions of a stakeholder, with varying opinions about the communities that should be included in governance. They do agree, however, that these discussions are paramount to ensure beneficence and justice, tenets of bioethical thought, for those involved. 

An underlying reason for these concerns is that, should human germline genome editing become widely available in the future, the cost of these therapies might restrict access to certain privileged populations.

“I don’t think it’s far-fetched to say that there’s institutionalized racism that goes on around access to this technology, the democratization and self-governance of it,” said Keolu Fox, a UC San Diego scholar who studies the anthropology of natural selection from a genomics perspective. Fox focused his discussion on indigenous populations when addressing the issue of autonomy in governance of germline genome editing [26]. 

“If we don’t put indigenous people or vulnerable populations in the driver’s seat so that they can really think about the potential applications of this type of technology, self-governance, and how to create intellectual property that has a circular economy that goes back to their community,” Fox said, “that is continued colonialism in 2020.”

Indeed, marginalized communities have experienced the evil that genetics can be used to justify, and millions of lives have been lost throughout human history to ideologies emphasizing genetic purity like eugenics and Nazism. 

“We know that history with genetics is wrought with a lot of wrongdoings and also good intentions that can go wrong, and so there’s a community distrust [of germline editing],” said Billie Liangolou, a UC San Francisco (UCSF) Benioff Children’s Hospital genetic counselor, during a panel on stakeholders that included Fox. Liangolou works with expecting mothers, guiding them through the challenges associated with difficult genetic diagnoses during pregnancy [26].

Others agree that the communities affected most by human germline genome editing should be at the forefront of decision-making about this emerging technology. Sharon Begley, a senior science writer at STAT News, told the conference audience that a mother with a genetic disease once asked her if she could “just change my little drop of the human gene pool so that my children don’t have this terrible thing that I have” [26].

This question, frequently echoed throughout society by other prospective parents, reflects the present-day interest in human germline genome editing technologies, interest that will likely continue to grow as further research on human embryos continues.

In an opinion published by STAT News, Ethan Weiss, a cardiologist and associate professor of medicine at UCSF, acknowledges the concerns of parents faced with these decisions [27]. His daughter, Ruthie, has oculocutaneous albinism, a rare genetic disorder characterized by mutations in the OCA2 gene, which is involved in producing melanin. Necessary for normally functioning vision, melanin is a pigment found in the eyes [28].

Weiss and his partner “believe that had we learned our unborn child had oculocutaneous albinism, Ruthie would not be here today. She would have been filtered out as an embryo or terminated,” he said.

But, in the end, Weiss offers up a cautionary message to readers, encouraging people to “think hard” about the potential effects of human germline genome editing. 

“We know that Ruthie’s presence in this world makes it a better, kinder, more considerate, more patient, and more humane place,” Weiss said. “It is not hard, then, to see that these new technologies bring risk that the world will be less kind, less compassionate, and less patient when there are fewer children like Ruthie. And the kids who inevitably end up with oculocutaneous albinism or other rare diseases will be even less ‘normal’ than they are today.”

Weiss’ warning is underscored by disability rights scholars who say that treating genetic disorders with CRISPR or other germline editing technologies could lead to heightened focus on those who continue to live with these disabilities. In an interview with Katie Hasson of the Center for Genetics and Society, located in Berkeley, Jackie Leach Scully commented on the stigmatization that disabled people might face in a world where germline editing is regularly practiced [29].

“Since only a minority of disability is genetic, even if genome editing eventually becomes a safe and routine technology it won’t eradicate disability,” said Scully, professor of bioethics at the University of New South Wales in Australia. “The concern then would be about the social effects of [heritable genome editing] for people with non-genetic disabilities, and the context that such changes would create for them.” 

Others worry about how to define the boundary between the prevention of genetic diseases and the enhancement of desirable traits—and what this means for the decisions a germline editing governing body would have to make about people’s value in society. Emily Beitiks, associate director of the Paul K. Longmore Institute on Disability at San Francisco State University, is among the community of experts who have raised such concerns [30].

 “Knowing that these choices are being made in a deeply ableist culture,” said Beitiks in an article posted on the Center for Genetics and Society’s blog [30], “illustrates how hard it would be to draw lines about what genetic diseases ‘we’ agree to engineer out of the gene pool and which are allowed to stay.”

Religious leaders have also weighed in on the ethics of human germline genome editing. Father Joseph Tham, who has previously published work on what he calls “the secularization of bioethics,” presented his views on the role of religion in this debate about bioethics at the conference [26].

“Many people in the world belong to some kind of religious tradition, and I think it would be a shame if religion is not a part of this conversation,” said Tham, professor at Regina Apostolorum Pontifical University’s School of Bioethics.

Tham explained that the church already disapproves of IVF techniques, let alone human germline editing, “because in some way it deforms the whole sense of the human sexual act.”

Islamic perspectives on germline editing differ. In a paper published last year, Mohammed Ghaly, one of the conference panelists, discussed how the Islamic religious tradition informs perspectives on human genome editing in the Muslim world [31].

“The mainstream position among Muslim scholars is that before embryos are implanted in the uterus, they do not have the moral status of a human being,” said Ghaly, professor of Islam and biomedical ethics at Hamad Bin Khalifa University. “That is why the scholars find it unproblematic to use them for conducting research with the aim of producing beneficial knowledge.”

Where Muslim religious scholars draw the line, Ghaly says, is at the applications of human germline genome editing, not research about it. Issues regarding the safety and effectiveness of germline editing make its current use in viable human embryos largely untenable, according to the majority of religious scholars [31].

The unfolding, back-and-forth debate about who and how to design policies guiding human germline genome editing continues to rage on, but there is little doubt about consensus on one point. For a technology with effects as far-reaching as this one, time is of the essence.

 

References

  1. Scientist who claims to have made gene-edited babies speaks in Hong Kong . 27 Nov 2018, 36:04 minutes. Global News; [accessed 2 May 2020]. https://youtu.be/0jILo9y71s0.
  2. Cyranoski D. 2018. CRISPR-baby scientist fails to satisfy critics. Nature. 564 (7734): 13-14. 
  3. Regalado A. 2019. China’s CRISPR babies: Read exclusive excerpts from the unseen original research. Cambridge (MA): MIT Technology Review; [accessed 2 May 2020]. https://www.technologyreview.com/2019/12/03/131752/chinas-crispr-babies-read-exclusive-excerpts-he-jiankui-paper/.
  4. Genetic Engineering Will Change Everything Forever – CRISPR . 10 Aug 2016, 16:03 minutes. Kurzgesagt – In a Nutshell; [accessed 2 May 2020]. https://youtu.be/jAhjPd4uNFY. 
  5. Doudna J. Editing the Code of Life: The Future of Genome Editing [lecture]. 21 Feb 2019. Endowed Elberg Series. Berkeley (CA): Institute for International Studies. https://youtu.be/9Yblg9wDHZA. 
  6. Haft DH, Selengut J, Mongodin EF, Nelson KE. 2005. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol. 1 (6): e60. [about 10 pages].
  7. Makarova KS, Koonin EV. 2015. Annotation and Classification of CRISPR-Cas Systems. Methods Mol Biol. 1311: 47-75.
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  9. Enzmann B. 2019. CRISPR Editing is All About DNA Repair Mechanisms. Redwood City (CA): Synthego; [accessed 2 May 2020]. https://www.synthego.com/blog/crispr-dna-repair-pathways.
  10. Normile D. 2019. Chinese scientist who produced genetically altered babies sentenced to 3 years in jail. Science; [accessed 2 May 2020]. https://www.sciencemag.org/news/2019/12/chinese-scientist-who-produced-genetically-altered-babies-sentenced-3-years-jail.
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  12. Osborne H. 2020. China confirms three gene edited babies were born through He Jiankui’s experiments. New York City (NY): Newsweek; [accessed 2 May 2020]. https://www.newsweek.com/china-third-gene-edited-baby-1480020.
  13. Cohen J. 2019. Embattled Russian scientist sharpens plans to create gene-edited babies. Science; [accessed 2 May 2020]. https://www.sciencemag.org/news/2019/10/embattled-russian-scientist-sharpens-plans-create-gene-edited-babies#.
  14. Zayner J. 2020. CRISPR babies scientist He Jiankui should not be villainized –– or headed to prison. Boston (MA): STAT News; [accessed 2 May 2020]. https://www.statnews.com/2020/01/02/crispr-babies-scientist-he-jiankui-should-not-be-villainized/.
  15. Baer N. 2020. Covid-19 is scary. Could a rogue scientist use CRISPR to conjure another pandemic? Boston (MA): STAT News; [accessed 2 May 2020]. https://www.statnews.com/2020/03/26/could-rogue-scientist-use-crispr-create-pandemic/.
  16. Wang H, Yang H. 2019. Gene-edited babies: What went wrong and what could go wrong. PLoS Biol 17 (4): e3000224. [about 5 pages].
  17. About Lulu and Nana: Twin Girls Born Healthy After Gene Surgery As Single-Cell Embryos . 25 Nov 2018, 4:43 minutes. The He Lab; [accessed 2 May 2020]. https://youtu.be/th0vnOmFltc.
  18. Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, Saragosti S, Lapouméroulie C, Cognaux J, Forceille C, et al. 1996. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature. 382 (6593): 722–5.
  19. Marmor M, Sheppard HW, Donnell D, Bozeman S, Celum C, Buchbinder S, Koblin B, Seage GR. 2001. Homozygous and heterozygous CCR5-Delta32 genotypes are associated with resistance to HIV infection. J Acquir Immune Defic Syndr. 27 (5): 472–81.
  20. Lopalco L. 2010. CCR5: From Natural Resistance to a New Anti-HIV Strategy. Viruses. 2 (2): 574–600.
  21. Martinson JJ, Chapman NH, Rees DC, Liu YT, Clegg JB. 1997. Global distribution of the CCR5 gene 32-base-pair deletion. Nat Genet. 16 (1): 100–3. 
  22. Zhang C, Fu S, Xue Y, Wang Q, Huang X, Wang B, Liu A, Ma L, Yu Y, Shi R, et al. 2002. Distribution of the CCR5 gene 32-basepair deletion in 11 Chinese populations. Anthropol Anz. 60 (3): 267–71.
  23. Sofia M. Yep. They Injected CRISPR Into an Eyeball . 19 May 2020, 8:43 minutes. NPR Short Wave; [accessed 2 May 2020]. https://www.npr.org/2020/03/18/yep-they-injected-crispr-into-an-eyeball.
  24. Zafer M, Horvath H, Mmeje O, van der Poel S, Semprini AE, Rutherford G, Brown J. 2016. Effectiveness of semen washing to prevent human immunodeficiency virus (HIV) transmission and assist pregnancy in HIV-discordant couples: a systematic review and meta-analysis. Fertil Steril. 105 (3): 645–55.
  25. Halpern J, O’Hara S, Doxzen K, Witkowsky L, Owen A. 2019. Societal and Ethical Impacts of Germline Genome Editing: How Can We Secure Human Rights? The CRISPR Journal. 2 (5): 293-298. 
  26. CRISPR Consensus? Public debate and the future of genome editing in human reproduction [conference]. 26 Oct 2019. Berkeley, CA: Innovative Genomics Institute. https://youtu.be/SFrKjItaWGc.
  27. Weiss E. 2020. Should ‘broken’ genes be fixed? My daughter changed the way I think about that question. Boston (MA): STAT News; [accessed 2 May 2020]. https://www.statnews.com/2020/02/21/should-broken-genes-be-fixed-my-daughter-changed-the-way-i-think-about-that-question/.
  28. Grønskov K, Ek J, Brondum-Nielsen K. 2007. Oculocutaneous albinism. Orphanet J Rare Dis. 2 (43). [about 8 pages]. 
  29. Hasson K. 2019. Illness or Identity? A Disability Rights Scholar Comments on the Plan to Use CRISPR to Prevent Deafness. Berkeley (CA): Center for Genetics and Society; [accessed 2 May 2020]. https://www.geneticsandsociety.org/biopolitical-times/illness-or-identity-disability-rights-scholar-comments-plan-use-crispr-prevent.
  30. Beitiks E. 5 Reasons Why We Need People with Disabilities in The CRISPR Debates. San Francisco (CA): Paul K. Longmore Institute on Disability; [accessed 2 May 2020]. https://longmoreinstitute.sfsu.edu/5-reasons-why-we-need-people-disabilities-crispr-debates.
  31. Ghaly M. 2019. Islamic Ethical Perspectives on Human Genome Editing. Issues in Science and Technology. 35 (3): 45-48.

Gene editing invasive species out of New Zealand

March 6, 2020

By Jessie Lau, Biochemistry and Molecular Biology ‘20

Authors Note: Since the advent of Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR Associated Protein 9 (CRISPR/Cas9) discovery and biotechnological breakthroughs thereafter, this revolutionary application has been primarily focused on human health, particularly fostering solutions to numerous debilitating ailments. However, the general public has offered little attention towards the use of this engineering feat in a broader ecological system. Upon watching the new Netflix original Unnatural Selection, discussion of considering the use of CRISPR/Cas9 in New Zealand’s effort to completely eradicate invasive species piqued my interest. The following article is an exploration of CRISPR/Cas9 prospect into New Zealand’s bold environmental pursuit and its potential ecological impact.

 

Abstract

Since it has become feasible to cross oceans to reach unforeseeable land masses, invasive alien species (IAS) are an increasing threat to international biodiversity. Moreover, no other region faces as great of a peril as New Zealand (NZ), which holds the record as the nation with the highest survival rate of threatened avifauna (birds of a particular region) species [5]. In 2012, New Zealand physicist Sir Paul Callaghan introduced a large-scale eradication program to permanently remove eight invasive mammalian predators (rodents: Rattus rattus, Rattus norvegicus, Rattus exulans, Mus musculus; mustelids: Mustela furo, Mustela erminea, Mustela nivalis; and the common brushtail possum: Trichosurus vulpecula) [5]. Four years after this grand proposal, the NZ government committed to a national challenge titled “Predator Free 2050” (PF 2050) to pursue this audacious goal. 

 

Introduction

Approximately 85 million years ago, NZ was one of the first landmasses to split from the supercontinent Gondwana and as it shifted away, it did not carry mammals until bats flew and aquatic species swam to this island. [7]. With the introduction of rodent species initially through Polynesian settlement some 750 years ago and thereafter European seafaring ventures, the endemic species of NZ have been left vulnerable to novel predators. Consequently, at least 51 native bird species that have evolved adaptive skills of remaining close to the ground have been unsuccessful at surviving alongside these rodent predator species [4]. These invasive omnivorous rodents prey on birds, eggs, seeds, snails, lizards, and fruits. As a result, their varied diets prompt competition with the native fauna, further placing pressure on their vulnerable survival [7]. Despite CRISPR/Cas9 modifications offering the greatest potential in gene drive to eliminate these unwanted predators, several techniques implemented to control these invasive populations,  such as pesticides, trapping innovations, and biological factors have given favorable results.  

 

Past Successes and Future Endeavors 

For decades, the NZ government has pursued eradication initiatives to permanently eliminate foreign invasive species on small local islands. Through concerted efforts, several of these projects have proven successful in the restoration of the natural biodiversity this island nation boasts. 

In June of 2009, the NZ Department of Conservation (DOC) undertook a multi-action plan to simultaneously eradicate rodent, rabbit, stout, hedgehog, and cat species in Rangitoto and Motutapu islands. After three years of aerial dispersion of anticoagulant Pestoff 20RTM combined with trapping and indicator dogs, the island witnessed total elimination of the islands’ stoats and four rodent species; declination of rabbit and hedgehog population by 96%; and over 50% reduction in cats [7]. 

Despite these successful feats, the invasive species fecundity and ability to adapt to these challenges still present an overwhelming challenge to reach the goal of complete eradication. Recently, more direct approaches delving into novel genetic inheritance techniques have been explored to serve as a potential permanent solution. Termed the“Trojan Female Technique” (TFT), the method makes use of the correlation of sperm fitness dependence on the abundance of mitochondrial DNA (mtDNA) [2]. Healthy sperm is dependent on sufficient mitochondrial level for energy production to manage motility and fertilization. For example, experiments conducted on Drosophila melanogaster supports the causation of reduced spermatogenesis and sperm maturation due to induced mutations in cytochrome mitochondrial gene [9]. Contrasting female eggs, the asymmetric greater dependence of sperm on mtDNA for normal functionality results in only male populations to be the sole source of target. Induction of mitochondrial mutations in females to compromise total sperm viability in future male progenies will serve as an effective control to population growth. 

Although seemingly promising, TFT is not guaranteed to completely eradicate propagation for several reasons. For starters, males with impaired fertility can still provide sufficient sperm count to fertilize eggs on a population-based scale. Furthermore, in circumstances where females do receive nonviable sperm count, they can still seek adequate functioning sperm through matings with other males. On a larger scale, should the mutation pervade, selection pressures could still inadvertently choose for nuclear modifications to make up for the mitochondrial defects [2]. These flaws raise the need for more pervasive and permanent resolutions.

 

Daisy Chain CRISPR Gene Drive

The recent biochemical breakthrough underpinning the ability to effectively and precisely modify genes with CRISPR/Cas9 has allowed for potential biotechnology to boom in the realm of ecology. The simple generation of a short RNA sequence into a virus or bacterium to serve as a vector, guides the cutting mechanism of Cas9 to specific regions in the genome to be excised, prompting for these double stranded breaks to be fixed through DNA repair mechanisms. While these fixtures can potentially repair the gene, it can also raise the possibility for the gene to be disabled, introduce a new function, or create an unforeseen mutation. Given the right specific targeting in the germ line, this approach houses the innovation for exterminating entire species through gene drive [6].

The mechanism behind gene drive overthrows the traditional Mendelian sexual reproduction concept of proportional contribution from both the male and female parent. The power comes from the ability of one genetically modified (GM) contributor encoding for the ‘gene drive’ to cut the other set of chromosomes lacking these genes and replace this excision with a self-replicating copy. In effect, this divisive modification would push for an otherwise heterozygous offspring from a wild-type mating with a GM partner to become homozygous for certain genes to be carried on and propagated by future generations. Given that these genetic alterations do not affect the fitness of the organism, dissemination of 1% of the population with CRISPR modified genes can lead to 99% of the local population carrying the genetic indicator in as little as nine generations (The use of gene editing to create gene drives for pest control in New Zealand).

Such a unilateral approach poses political and ethical challenges amongst neighboring nations with diverging ecological approaches to confront pest control. Should this pervasive gene drive program reach beyond its intended border, great difficulty would arise in maintaining this ecological enclosure. For example, possum is on the list of invasive species in NZ while just 2,500 miles west, its neighbor Australia keeps this species of marsupials under protection. As such, scientists have devised a simple model to localize gene alterations, coined The Daisy Chain.

Unlike the original gene drive method in which all components necessary for transformation (CRISPR, edited DNA, and guide RNAs) are provided on the same chromosome, Daisy Chain provides a self-exhaustive means of guaranteeing genetic edits. This tool is designed so that  each component required for genetic alteration is dependent on the presence of a different element upstream on the gene found on the same locus to be activated [8]. The most downstream portion of this chain contains the “load” of engineered dominant lethal genes preventing reproduction, which will be promoted to higher frequency in the population within several generations. For instance, should an engineered allele contain three elements A, B, and C, element C would render element B to drive, which will in turn cause element A carrying the final load to drive. The initial element (C in this case) does not actually drive, thus is restricted by the number of altered individuals released into the wild and will be lost via natural selection over time. During initial implementation, the presence of C will increase B in abundance, but B will eventually decline and finally disappear as C is lost in the population. The rapid rise in abundance of B will also cause A to increase in frequency within the local population; however, with the decrease of B, A would not be driven and will ultimately vanish as well [1]. Using MIT Professor Esvelt’s analogy, “… the elements of a daisy drive system are similar to booster stages of a genetic rocket: those at the bottom of the base of the daisy-chain help life the payload until they run out of fuel and are successively lost.”

 

Challenges with Daisy Chain CRISPR Gene Drive

CRISPR/Cas9 technology’s ability to potentially alter these invasive species’ fecundity provides an avenue of pursuing NZ’s goal of PF 2050. Despite the developed understanding of how to carry out this plan, scientists in NZ are still working to piece together the genomes of stoats and possums in order to understand where to properly facilitate the engineered RNA sequence. Other barriers that must be acknowledged are the unprecedented approach to genetically modify marsupials and the difficulty of implanting hundreds to thousands of oocytes to be dispersed amongst their population. 

Beyond these known difficulties, scientists still tread in unknown terrains pertaining to whether these mutations can have pernicious effects in the survival, health, and reproductive success in propagating these mutations within their populations. Further exploration into the development of these modifications, and the potential impacts they can have on these animals, must be investigated on model organisms prior to widespread use. 

Of the eight listed mammalian species vied to be permanently eradicated from NZ, Mus musculus holds the most promise, given the extensive knowledge of the Mus musculus genome. With the help of scientists outside of New Zealand, joining in on the efforts to identify which germline gene to focus on, this project has received international attention.

Although the daisy drive provides promising potential, research collaborators at MIT and Harvard have identified a possible risk of, “… DNA encoding a drive component from one element to another, thereby creating a ‘daisy necklace’ capable of a global drive” [1]. Due to this rare recombinatory event arising from the similarity of DNA sequences, these investigators have looked into circumventing the problem by creating numerous alternatives to CRISPR components and selecting the model with the greatest diversity. 

 

Conclusion

From their renowned aviary to reptilian species, New Zealand’s islandic geographical region houses some of the most biodiverse fauna known to man. The arrival of human settlement has introduced predatory species, causing endemic species to experience extinction at concerning rates [4]. With the purpose of preserving their unique remaining diversity, New Zealand has committed to concerted efforts of varying methods to eradicate these invasive vertebrate pests. Investigation into genetic modifications can provide for more expansive and thorough techniques to eliminate these human introduced pests and allow for these endangered species to thrive once again. By further exploring daisy chain CRISPR/Cas9, this effort can be genetically inherited by offspring, allowing for nature to carry out this effort. As opposed to continued efforts of targeting each individual one by one, conservation ecologists can borrow from molecular biologist’s toolkit to revolutionize the means of pursuing pest control and perhaps even pave the road for future endeavors with similar pursuits. 

 

References

  1. Esvelt, Kevin M. “Daisy Drives.” Sculpting Evolution, www.sculptingevolution.org/daisydrives.
  2. Gemmell, Neil J., et al. “The Trojan Female Technique: a Novel, Effective and Humane Approach for Pest Population Control.” Proceedings of the Royal Society B: Biological Sciences, vol. 280, no. 1773, 2013, pp. 1–6., doi:10.1098/rspb.2013.2549.
  3. Griffiths, Richard, et al. “Successful Eradication of Invasive Vertebrates on Rangitoto and Motutapu Islands, New Zealand.” Biological Invasions, vol. 17, no. 5, 2014, pp. 1355–1369., doi:10.1007/s10530-014-0798-7.
  4. Owens, Brian. “The Big Cull: Can New Zealand Pull off an Audacious Plan to Get Rid of Invasive Predators by 2050?” Nature, vol. 541, 12 Jan. 2017, pp. 148–150.
  5. Russell, James C., John G. Innes, Philip H. Brown, and Andrea E. Byrom. “Predator-Free New Zealand: Conservation Country.” BioScience 65, no. 5 (October 2015): 520–25. https://doi.org/10.1093/biosci/biv012.
  6. Saey, Tina Hesman. “Explainer: How CRISPR Works.” Science News for Students, 4 Dec. 2017, www.sciencenewsforstudents.org/article/explainer-how-crispr-works.
  7. “Why Predator Free 2050?” Department of Conservation. Accessed November 18, 2019. http://www.doc.govt.nz/nature/pests-and-threats/predator-free-2050/why-predator-free-2050/.
  8. Dearden, Peter K., et al. “The Potential for the Use of Gene Drives for Pest Control in New Zealand: a Perspective.” Journal of the Royal Society of New Zealand, vol. 48, no. 4, 2017, pp. 225–244., doi:10.1080/03036758.2017.1385030.
  9. Wolff, Jonci N., et al. “Mitonuclear Interactions, MtDNA-Mediated Thermal Plasticity and Implications for the Trojan Female Technique for Pest Control.” Scientific Reports, vol. 6, no. 1, 2016, doi:10.1038/srep30016.
  10. Min, John, Jason Olejarz, Joanna Buchthal, Alejandro Chavez, Andrea L. Smidler, Erika A. DeBenedictis, George M. Church, Martin A. Nowak, Kevin M. Esvelt, and Charleston Noble. “Daisy-Chain Gene Drives for the Alteration of Local Populations.” PNAS. National Academy of Sciences, April 23, 2019. https://www.pnas.org/content/116/17/8275.

CRISPR/HDR Platform Allows for the Production of Monoclonal Antibodies with the Constant Region of Choice

November 22, 2019

By Sharon Yang, Cell Biology, ‘20

Author’s Note: I first came across an article talking about this new innovation on Science X. Having worked with hybridomas and antibodies through various internships, I was deeply intrigued by this discovery and secured an original paper to learn more about its potential applications. Because of the revolutionizing usage of antibodies in the medical field, it is vital to understand how this finding will facilitate antibody-based therapies in clinical research.  

 

Introduction

Since the discovery of antibodies and their applications in therapeutics, many diseases once deemed incurable now have a treatment, if not a cure. Antibodies are proteins that recognize and bind to specific antigens (proteins that are considered “foreign” to the body). The immune system recognizes this antibody-antigen complex and removes the foreign substance from the body. Monoclonal antibodies (mAbs) are specific for one type of antigen and are produced using hybridomas, immortal cell lines that secrete only one type of antibody. The specificity of a mAb is determined by its antigen binding variable region. Though the variable region is of critical importance, the constant region (also known as the Fc region) is also essential to the therapeutic efficacy of mAbs. The Fc region has many different variants, called isotypes. Each isotype has its own unique function in making the immune system respond in different ways. After an antibody binds to an antigen by its variable region, the Fc region of the antibody elicits a response from the immune system, which serves as the basis for antibody-based therapeutics.

A recent study conducted in the summer of 2019 by Schoot and colleagues demonstrates how the use of genetic engineering on hybridomas can modify the Fc region of mAbs to that of a different species, isotype, or format. This new versatile platform grants ease of production of monoclonal antibodies that have different constant regions but retain the same variable regions. 

The research team utilized a one-step clustered regularly interspaced short palindromic repeat (CRISPR)/homology-directed repair (HDR) technique to create a recombinant hybridoma that secretes a mAb in the Fc format of choice — a highly attractive alternative to the conventional recombinant production methods that were often time-consuming, challenging, and expensive.

As the team emphasizes, “[CRISPR/HDR] is a simple alternative approach requiring a single electroporation step to obtain an unlimited source of target antibody in the isotype format of choice” (1). Through using CRISPR/HDR, the team was able to seamlessly generate monovalent Fab’ fragments and a panel of different isotypes for the same monoclonal antibody.

 

CRISPR/Cas9 and Homology-Directed Repair

In their genetic engineering method, the researchers took advantage of an ancient bacterial immunity mechanism: the CRISPR/Cas9 system. When a bacteria is invaded by a virus, the bacteria stores snippets of viral DNA and creates segments of DNA called CRISPR arrays. When a virus with the same DNA segment attacks again, the bacteria creates RNA from the CRISPR arrays to target the virus; the RNA is called the guide RNA. The nuclease protein Cas9 is used to cut the DNA apart at a very specific site determined by the guide RNA, disabling the virus. CRISPR/Cas9 works in a similar fashion in the lab. Scientists create a guide RNA that binds to Cas9, which then targets a specific site on the DNA to be cut (2).

When CRISPR/Cas9 cuts DNA, it induces a double-strand break (DSB). Homology-directed repair (HDR) occurs when the intact donor strand contains high sequence homology to the damaged DNA strand. Through HDR, scientists can integrate a sequence or gene of their liking into the genome, which Schoot and colleagues perform in their study (3).  

 

The Generation of Fab’ Fragments

The fragment antigen-binding (Fab’) is a region on the antibody that binds to the antigen. It consists of a single heavy chain and light chain. To create a Fab’ fragment-secreting hybridoma using CRISPR/HDR, the team selected NLDC-145, a hybridoma clone that secretes mAbs of rat IgG2a (rIgG2a) isotype. The antigen of rIgG2a is DEC205, an endocytic receptor found on immune cells. The team electroporated NLDC-145 cells with Cas9 and an appropriate guide RNA to induce double-strand breaks at the hinge region; to repair the double-strand break, they designed an HDR Fab’ donor construct for homology-directed repair. The HDR Fab’ donor construct also inserts specific tags onto the protein, allowing for easy purification of the Fab’ fragment. 

To test secretion of the Fab’ fragment, they stained JAWSII, a DEC205-expressing cell line, with the supernatants of NLDC-145 clones that had undergone CRISPR/HDR. Flow cytometry assays showed that a large portion of Fab’-secreting hybridomas were successfully created. Further assays showed that the secreted Fab’ fragments retained their binding capabilities. It is worth noting that the researchers also used the same strategy to convert other hybridoma lines to become recombinant, Fab’-producing lines, with similar success; this demonstrates that this engineering technique is flexible and not just limited to one cell line (1).

 

The Generation of Isotype Panels

In a similar manner to creating monoclonal Fab’-generating hybridomas, the team also used the one-step CRISPR/HDR technique to create hybridomas capable of producing a wide array of isotype variants for the same mAb. This time, the cell line subject was hybridoma MIH5, which secretes monoclonal rIgG2a that targets mouse PD-L1, an immune checkpoint protein. The goal was to make clones of MIH5 to each produce one isotype of the chimeric (having both rat and mouse-related parts) monoclonal antibodies: mIgG1, mIgG2a, mIgG2b, mIgG3, mIgA, and a mutant form of mIgG2a (mIgG2asilent).

MIH5 cells were cotransfected (introduced with DNA) with a Cas9 vector containing the appropriate guide RNA and a construct from a panel of isotype HDR donor constructs (each isotype had its own unique HDR donor construct). Following knock-in integration, flow cytometry analysis showed that the engineered chimeric mAbs were successfully secreted. Thus, the creation of recombinant hybridomas for a panel of isotypes was successfully engineered (1). This invention allows for the creation of monoclonal antibodies with different Fc regions, providing researchers an easy way to “customize” their antibodies to elicit a specific response from the immune system. Researchers may choose which isotype variant they want on their antibody, which is fully dependent on their target (antigen) of interest and how the immune system behaves towards it. This has vast potential in antibody-based therapeutics, in that this system can be used for the optimization of potential drugs to become more potent and dynamic.

 

Biochemical Applications

To test the functional capability of isotype-switched mAbs, Schoot and colleagues tested the antibodies’ capability to induce an important immune mechanism: antibody-dependent cellular cytotoxicity (ADCC). In order to test ADCC in vitro, mouse colon adenocarcinoma cells were labeled with chromium-51, and then taken in by MIH5 Fc isotype variants. After adding whole blood, they measured chromium-51 release. On the other hand, B cell depletion by MIH5 Fc variants was used to measure ADCC in in vivo experiments. Analyses of these studies show that chimeric mAbs created by CRISPR/HDR hybridomas have the same biochemical and immune effector characteristics as their recombinant and naturally occurring counterparts (1). Something to highlight is that instead of treading through the laborious process of producing  recombinant antibodies in the conventional way (often consisting of multiple rounds of optimized sequencing, cloning, transfection), this one-step mechanism grants smooth and rapid generation of recombinant antibodies that perform their expected functions (1).

 

Conclusion

The ability to create monoclonal antibodies with the freedom to choose what goes on their constant regions possess many applications in the vast field of medicine and engineering. Being able to construct a very specific monoclonal antibody (the engineering element) that stimulates the immune system in a certain, beneficial way (the medical component) intertwines the two fields together to propel us closer towards treating diseases more efficiently and effectively. This system also represents an optimized version of recombinant engineering, which saves valuable time and funds that can be used towards conducting further studies. A simple, yet powerful and flexible approach, this versatile CRISPR/HDR platform aims to facilitate antibody engineering and research for the scientific community, and is accelerating the rate at which new clinical trials can be performed.

 

References

  1. Schoot, J. M. V. D. et al. Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering. Science Advances 5, (2019).
  2. Ran, F Ann et al. “Genome engineering using the CRISPR-Cas9 system.” Nature protocols vol. 8,11 (2013): 2281-2308. doi:10.1038/nprot.2013.143
  3. Cortez, Chari. “CRISPR 101: Homology Directed Repair.” Addgene Blog, Addgene, 12 Mar. 2015, blog.addgene.org/crispr-101-homology-directed-repair.

CRISPR: Are We Ready For It?

May 11, 2019

By Tannavee Kumar, Genetics and Genomics, ’20

Author’s Note: When I found out that CRISPR was used for the first time on human embryos that were fully brought to term, I was pretty surprised that such a new technology with numerous unknowns was being used on the germline. I was interested in understanding the reasoning for such an experiment and what may come out of it in the long run. To some, CRISPR may seem like a far off technology that could be applied to humans in the distant future. However, the experiment on the twin fetuses that went through this “genetic surgery” proves that CRISPR is happening now and is likely to stay.  

But first, how does CRISPR work?

CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats, is a genome editing technology that is cheaper, faster, more accurate, and more efficient than other existing genome editing methods [1]. The CRISPR-Cas9 system was initially adapted from the prokaryotic defense system, where CRISPRs are found in both archaea and bacteria [5]. CRISPR arrays serve as an integral part of the immune system by consisting of repeating sequences of genetic code which are interrupted by “spacer” sequences — residues of genetic code from previously attacking bacteriophages [1,5]. When the viruses attack again, the bacteria transcribe CRISPR RNAs (crRNAs) from the “spacer” regions in the CRISPR arrays to latch onto the viral DNA, where Cas9 —  an enzyme that recognizes the crRNA — will cut the DNA, causing the knockout to cease the progression of the attack [5].

In the lab, the CRISPR system works in a similar manner. Researchers can also utilize various other enzymes to cleave the unwanted DNA, one such being Cpf1. Where CRISPR-Cas9 would produce blunt ends — a complex where the DNA is cut at the same location on each strand — CRISPR-Cpf1 cuts DNA at different locations, thus creating short single-stranded overhangs [5]. These overhangs, otherwise called sticky ends, can help make the insertion of new DNA more stringent, accurate, and of the correct orientation. Various enzymes also recognize different areas on the DNA; this differentiation allows researchers to allocate enzymes to their area of study [5]. Once the DNA is cut, segments of interest can be inserted or deleted by using the cell’s mechanism of homology-directed repair (HDR) and non-homologous-end joining (NHEJ) [1, 6].

Scientists hope to expand their findings pertaining to CRISPR on a variety of issues, especially single-gene disorders such as hemophilia, cystic fibrosis, and sickle cell disease. In the long term, CRISPR also shows promise in preventing or treating cancer, heart disease, mental illness, and human immunodeficiency virus (HIV) infection [1]. However, these findings and advancements in application come with various ethical concerns. As of now, most changes introduced with genome editing are limited to somatic cells. This ensures that changes are restricted to few tissues and cannot be passed down from one generation to another; however, changes made to germline cells, or even to embryos, can be passed down to future generations. Since this area is still relatively new and unknown, there is not yet enough information about the accuracy and long-lasting impact. Off-target effects could prove disastrous as there would be an entirely new set of unforeseen consequences. Moreover, even at the right target, genes interact in a complicated matter; changes at one site could have implications in a completely different area. Multiple unknowns coupled with long term consequences led to a tremendous outcry after it was revealed that two twin girls were born after having changes made at their embryonic stage.

The Trial  

While He Jiankui has not published a paper describing the methodology and details on his experiment, he released a promotional video for his study on The He Lab YouTube channel on November 25, 2018. He describes how because “Mark” is HIV positive, he and his wife “Grace” were concerned that their children would potentially be positive as well [2]. This made them viable candidates for the experiment. He Jiankui mentions that along with fertilizing Grace’s egg with Mark’s sperm, the researchers also “sent a little bit of protein and instructions” that would do a “gene surgery” to remove the doorway in which HIV enters to “infect people” [2].  In the video, He claims that the team considered the surgery to be a success, as confirmed by genome sequencing [2]. Lastly, twin embryos were implanted via regular IVF “with one difference” [2].

This resulted in the first “CRISPR babies”: “Lulu” and “Nana.” He acknowledges that his actions will spark unparalleled controversy; however, he describes this path as no different than when in vitro fertilization (IVF) was first used to create Louise Joy Brown in 1977. According to the limited information published in Shenzhen HOME Women’s and Children’s Hospital ethics committee review application, mouse and monkey models were initially used to conduct “rigorous” early-test studies for the CCR5 gene since it serves as a receptor that the latches onto a white blood cell [3, 4, 7].

The next step was embryos of model organisms similar to humans were examined after going through CRISPR-Cas9 gene editing [3]. Genetically edited embryonic stem cells were isolated to determine if they abnormally propagated and whether they differentiated after genetic testing [3]. To address off-target effects, two methods were used. Firstly, using high-fidelity Cas9 enzymes, He was able increase stringency and reduce off target effects [3]. Secondly, the best single guide RNA (sgRNA) was selected for which consists of the crRNA fused to the scaffold tracrRNA sequence that Cas nuclease binds to [7]. Using these critical steps, the CCR5 region was able to be deleted.

What is Next?

According to the Associated Press, He altered the embryos of seven couples, with the birth of only Lulu and Nana so far [9, 10]. In every case, the father was infected with the disease, and the mother was HIV-negative. He’s goal was to introduce an uncommon, natural genetic variation that would make it difficult for HIV to infect white blood cells [8]. By deleting the receptor, the apparent intent was not to reduce the chance of transmission from the father, since the likelihood naturally decreases when the sperm is washed before insemination during IVF. Rather, He stated that he wanted to reduce the chance of infection later on in life [8]. While He claims that he is against gene editing for enhancement purposes and that it should only be used for therapy, many question whether his actions prove hypocritical [2].  Many assert that He’s actions suggest that being born HIV susceptible is a disease state; however, common sense tells indicates otherwise, and that the lengths that He went through to reduce HIV susceptibility are not medically justified [8, 9].

The limited information suggests that He’s editing was incomplete, and that one of the twins is mosaic [9]. This would mean that only some cells have the silenced CCR5 gene, thus indicating that there is a significant likelihood that she would not be protected from HIV [9, 10]. Even with a successful deletion all around, certain strains of HIV can still enter cells through CXCR4, a protein [9]. When geneticists first identified people with a natural genetic variation consisting of non-working copies of CCR5, it was thought that they would be resistant to HIV without any repercussions [9].  However, later studies showed that deficiency in CCR5 meant that people were more susceptible to infections like West Nile virus, Japanese encephalitis, and more likely to die from influenza [9, 11]. While CRISPR can help spearhead numerous medical advancements, there is no doubt that many unknowns remain. Unintended consequences that the twins and other potential children of this study could face would prove that the progression of CRISPR applications for humans, especially for embryos, is premature.

References

  1. “What Are Genome Editing and CRISPR-Cas9? – Genetics Home Reference – NIH.” U.S. National Library of Medicine, National Institutes of Health. Web.
  2. He, Jiankui. About Lulu and Nana: Twin Girls Born Healthy After Gene Surgery As Single-Cell Embryos. YouTube, The He Lab, 25 Nov. 2018. Video.
  3. He, Jiankui. “Shenzhen HOME Women’s and Children’s Hospital Medical Ethics Committee Review Application.” Chinese Clinical Trial Registry , 8 Nov. 2018.
  4. He, Jiankui, and Jinzhou Qin. “Chinese Trial Registry: Trial Information.” Guangdong, CHina, 8 Nov. 2018.
  5. “Questions and Answers about CRISPR.” Broad Institute, MIT & Harvard University , 4 Aug. 2018. Web.
  6. “What Is the Difference between Non-Homologous End Joining (NHEJ) and Homology-Directed Repair (HDR)?” Web.
  7. “Full Stack Genome Engineering.” Synthetic Guide RNA for CRISPR Genome Editing | Synthego, SYNTHEGO. Web.
  8. Normille, Dennis. “CRISPR Bombshell: Chinese Researcher Claims to Have Created Gene-Edited Twins.” Science | AAAS, American Association for the Advancement of Science, 27 Nov. 2018. Web.
  9. Yong, Ed. “A Reckless and Needless Use of Gene Editing on Human Embryos.” The Atlantic, Atlantic Media Company, 27 Nov. 2018. Web.
  10. Marchione, Marilynn. “Chinese Researcher Claims First Gene-Edited Babies.” AP News, Associated Press, 26 Nov. 2018. Web.
  11. Falcon, A., et al. “CCR5 Deficiency Predisposes to Fatal Outcome in Influenza Virus Infection.” Journal of General Virology, vol. 96, no. 8, 2015, pp. 2074–2078., doi:10.1099/vir.0.000165.