In the last three years, the gene editing tool CRISPR-Cas9 has permeated nearly every crevice of the biological sciences and has revolutionized the way researchers think about the genetic code. However, the history behind CRISPR-Cas9 stretches much longer than three years, and it is proving to be a much more flexible platform than was ever imagined. At the same time, a few ethical controversies have been raised about the use of CRISPR-Cas9 in engineering human life, particularly the germline. As science moves into an era of personalized medicine and new approaches to cellular and gene therapies, CRISPR-Cas9 will undoubtedly solidify its place at the forefront of these exciting technologies. Following is an overview on the development and applications of CRISPR-Cas9 and the main ethical questions currently under discussion.
Development of CRISPR-Cas9:
Since the discovery of the role of DNA in the 1940s, the ability to manipulate the genome in a precise manner has been a dream of many researchers. However, CRISPR-Cas9 is only the latest breakthrough in a long line of tools designed to achieve this dream. In the 1990s, the first tools were developed to induce a double-stranded break at a specific place in the DNA molecule in living cells. Reengineering Zinc Fingers, a type of protein found in many organisms which is known to bind specific DNA sequences, allowed specific targeting and cutting of a locus. The damaged cell then uses its own innate repair mechanisms to fix the break, leading to one of two distinct results. First, and most often, the cell performs non-homologous end joining (NHEJ) which typically leads to extensive and unpredictable mutations. Because it is so error-prone, NHEJ is ideal for gene knockout research, in which a gene is disrupted in order to assess the resulting effect on the cell or organism. However, it is too inexact for more precise applications. As an alternative, a strand of “donor DNA” with sequences that match both sides of the cut site can be incorporated. In homology-directed repair (HDR), this donor DNA recombines with the cut DNA strands to be inserted into the genome. Researchers have used this method both to repair a mutation by providing the correct DNA sequence and to insert an entirely new gene into a specific host locus, such as a fluorescent protein.
Meanwhile, CRISPR-Cas9 was discovered in bacteria as an immune system against invading viral DNA. When a virus inserts itself into a bacterium, the bacterium recognizes it as foreign and incorporates a short segment of viral DNA into its own genome. The bacterium then produces RNA transcripts from this segment, which are used by the Cas9 protein to recognize foreign DNA. When the RNA transcript matches invading viral DNA, the Cas9 protein cuts both strands of the DNA, effectively degrading the virus’s DNA while protecting its own host DNA. The “CRISPR” in CRISPR-Cas9 refers to the bacterial immune system as a whole, and Cas9 is the protein itself which cuts the DNA. In early 2013, the CRISPR system was adapted for use in mammalian cells by making an artificial single-guide RNA (sgRNA) to match with the target sequence in the cell’s genome. While Zinc Fingers require a new protein to be made for each individual cut site, the Cas9 protein couples with any custom sgRNA, drastically reducing the time to design and complete a new edit and allowing largescale genetic screens to be performed. Additionally, CRISPR-Cas9 can be used to edit cells both in a dish (in vitro) and in full organisms such as mice (in vivo). But CRISPR-Cas9 is not perfect. Because the sgRNA targets 20 base pairs, similar sequences within the genome can result in off-target cuts. In rare cases, these cuts occur within another gene, leading to unintended effects. However, with over one trillion possible combinations of twenty A, C, T, and G bases, these off-target effects are rare, and many computational tools have been developed to screen the genome and provide “scores” for potential sgRNA targets. Additionally, the effectiveness of CRISPR-Cas9 to access the target site depends on variations in the target sequence itself, as DNA sequences with additional G and C base pairs are more tightly bound than those with additional A and T base pairs, and large segments of DNA are inaccessible due to binding to packaging proteins. A good score for an sgRNA incorporates all of these factors. An emerging area of research is mutating the Cas9 protein in order to increase its specificity or alter its function. For example, modifying the part of the protein responsible for cutting each strand does not disrupt the ability of Cas9 to bind to the sgRNA or to localize to the target sequence in the DNA. Such a change can stop the Cas9 from cutting one strand, causing a single-stranded “nick” in the DNA which can be exploited for enhanced specificity. Likewise, altering both cutting sites can completely eliminate cutting activity, known as a “nuclease-dead” Cas9, or dCas9. When dCas9 is localized to a gene, it physically impedes innate DNAto-RNA transcription, effectively silencing the gene. This can be an efficient method to understand the function of a gene without permanently altering its sequence. Transcriptional activators can also be conjugated to a dCas9, which results in gene transcription when using an sgRNA targeting a promoter region for a gene, even one not normally expressed.
Biomedical and Biotechnology Applications:
Empowered by the efficient gene editing tool of CRISPR-Cas9, biomedical researchers are now able to apply many new techniques that hold great promises in advancing human health. Gene insertion and deletion allows researchers to manipulate the expression of important proteins and metabolites in cells, while controlling gene expression permits researchers to better understand the function of these genes and the complex biology of the cell. Moreover, as a nearly universal gene editing tool, CRISPRCas9 greatly broadens the applicability of common model organisms and enhances the efficiency of engineering new model systems.
“CRISPR-Cas9 has enabled modeling of numerous diseases and processes, including cancer, neurodegeneration, and cardiovascular disease.”
Besides applying CRISPR-Cas9 for understanding the fundamental genetics of an organism, biomedical researchers use CRISPR-Cas9 gene editing as a powerful tool for understanding human diseases. By introducing genes known to be implicated in disease into cells in vitro, CRISPR-Cas9 serves as a powerful tool for disease modeling. For example, researchers use CRISPR-Cas9 in conjunction with human pluripotent stem cells to create organoids that express disease-specific genes in a culture dish. CRISPR-Cas9 has enabled modeling of numerous diseases and processes, including cancer, neurodegeneration, and cardiovascular disease. In addition to being able to observe and to understand the progression of disease through these models, CRISPR-Cas9 is also opening new avenues for the development of novel drugs. Pharmaceutical companies are testing the safety and efficacy of new drug candidates in these disease models, facilitating faster, more cost-effective, and safer drug development.
CRISPR-Cas9 is not only efficient in introducing disease genes into cell models in vitro, but it also holds great promise for the insertion of genes to correct deleterious mutations. The human genome consists of approximately 25,000 known genes, of which more than 3,000 are linked with diseases when mutated; these genes either lack necessary functionality or generate malfunctioning proteins and metabolites with deleterious effects. While conventional gene therapy aims to introduce additional copies of normal or ‘healthy’ genes, biomedical researchers have proposed that CRISPR-Cas9 is sufficiently efficacious, accurate, and adaptable to perform true therapeutic genome editing and fix a native locus. Dr. Timothy Kamp, a physicianscientist on campus, emphasizes that CRISPR-Cas9 has the potential to become a powerful therapeutic tool for treating many genetic diseases, such as muscular dystrophy and cystic fibrosis. Apart from editing human cells for biomedical applications, the adaptability of CRISPR-Cas9 enables scientists to edit cells of nearly all organisms, ranging from bacteria to simple animals to complex vertebrates. It is thus possible to harness these organisms for their desired properties in nearly any biomedical or biotechnology application. For example, CRISPR-Cas9 has been used in yeast and in plants to engineer biofuel and pharmaceutical production. Industrial companies have proposed the use of CRISPR-Cas9 in the food industry, with applications ranging from engineering the bacteria that convert milk into yogurt to engineering the dairy cows themselves.
Another revolutionary application of CRISPR-Cas9 is the gene drive. The gene drive increases the probability that an offspring will inherit a particular copy of an allele. Over a few generations, the gene drive system spreads the allele in the natural population. One key application of the gene drive is to eliminate pest-borne diseases such as malaria and dengue fever by targeting the disease vectors, namely mosquitos. Conventional gene engineering tools such as homologous recombination have very low efficiency in engineering the desired allele in mosquitoes. CRISPR-Cas9 allows feasible gene drive engineering, which has been effective in driving the spread of particular genes in laboratory animals such as fruit flies. Moreover, the gene drive has been proposed to reverse pesticide and herbicide resistance in pests and weeds in the agricultural industry and to reduce invasive species, aiding in the conservation of native and rare species.
Just like many other new technologies, there are serious concerns about CRISPRCas9 applications. As an efficient tool for editing the genome of almost any organism, the use of CRISPR-Cas9 has been criticized as scientific overreach. Critics have questioned both the safety of modifying human genomes for medical applications and the safety of genetically modified organisms in agricultural applications.
The overarching ethical question surrounding CRISPR-Cas9 is the extent to which scientists should manipulate nature. Since CRISPR-Cas9 makes gene editing relatively fast and efficient, thus reducing the technological barriers in creating new transgenic organisms, scientists today have more power to manipulate living organisms than ever before. For example, biologists recently proposed the use of the gene drive to fight the spread of Zika virus, which has been linked to thousands of severe neurological birth defects over the last year. Dr. Kate O’Connor-Giles, a biomedical researcher on campus who studies fruit fly genetics through CRISPRCas9, has indicated that the recent Zika outbreak could be a trigger for scientists to consider using the gene drive to combat mosquito-borne diseases. Dr. O’ConnorGiles suggested that there are more ethical complexities apart from the extent of the manipulation considered, and that the individuals involved in these processes are a crucial component. In her words, “Who should make that decision? Should scientists, bioethicists and citizens in Zikaafflicted areas have more say?” CRISPRCas9 itself does not give answers to these questions. Therefore, the ethical question of manipulating nature while utilizing gene editing and CRISPR-Cas9 is presently being debated.
Human genome editing by CRISPR-Cas9 is associated with additional ethical concerns. Dr. Krishanu Saha, a biomedical engineering researcher on campus, attended the International Summit on Human Gene Editing in December 2015. According to Dr. Saha, the major concern surrounding CRISPR-Cas9 is the editing of human germline cells and human embryos. Last year, a group of researchers at Sun Yat-sen University in China reportedly used CRISPR-Cas9 in unviable human embryos to demonstrate its ability to correct genetic diseases. However, the group concluded that there were too many unpredictable and variable effects to use CRISPR-Cas9 in viable embryos at this time. Furthermore, at the Francis Crick Institute in London, a group of researchers in February 2016 managed to obtain approval to edit viable human embryos using CRISPR-Cas9 to investigate early human development, under the stipulation that they may not keep the embryos longer than seven days. Importantly, neither of these studies have allowed genetically engineered embryos to implant and develop. Doing so would raise many additional ethical issues, such as the potential misuse of the technology for eugenics or enhancements, and the inequity of access to the technology for people of different socioeconomic statuses. Therefore, the consensus by the International Summit participants was that gene editing in human embryos and germline cells, while controversial in its own right, should be restricted to research purposes and these embryos should not be allowed to develop beyond early stages. The MIT Technological Review recently reported that gene editing was added to the list of threats posed by “weapons of mass destruction and proliferation” in the Annual Worldwide Threat Assessment Report of the U.S. intelligence community. The development of CRISPR-Cas9 as an efficient gene editing tool has made gene editing much easier because of the low cost and relative ease of use. Therefore, this concern arises from the potential for CRISPR-Cas9 to be used to create potentially harmful biological agents. However, the majority of the scientific community does not foresee these threats in the short term. The current regulations of gene editing are undergoing intensive revision to address the long term implications of the technology.
“As an effecient tool for editing the genome of almost any organism, the use of CRISPR-Cas9 has been criticized as scientific overreach.”
Despite such concerns, CRISPR-Cas9 has already enabled incredible scientific advancement and continues to hold great promise, and with continued development of CRISPR-Cas9, national and international regulations will be needed to address ethical concerns and the future of the field. The scientific community has been actively responding to some of the above concerns. For example, the National Academy of Sciences and the National Academy of Medicine collaborated on a gene-editing initiative to inform both researchers and technology and decisions surrounding its use. While risks will always exist, managing CRISPR-Cas9 through proper regulations and oversight will minimize these risks while permitting CRISPR-Cas9 to benefit society tremendously. CRISPRCas9 has expedited biotechnological advances within the last few years, enhancing understanding of organismal development and disease progression, and in the next few decades, ideally it will be commonplace to receive truly curative treatments for genetic diseases.
By Tianxiao Han and Ryan Prestil
We would like to thank Dr. Timothy Kamp, Dr. Kate O’Connor-Giles, and Dr. Krishanu Saha for the knowledgeable conversations during our interviews.
- Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014 Nov 28;346(6213):1258096.
- Klug A, Rhodes D. ‘Zinc fingers’: a novel protein motif for nucleic acid recognition. Trends in Biochemical Sciences. 1987 Jan 1;12:464-9.
- Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007 Mar 23;315(5819):1709-12.
- Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNAguided human genome engineering via Cas9. Science. 2013 Feb 15;339(6121):823-6.
- Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nature methods. 2013 Aug 1;10(8):741-3.
- Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. Highfrequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature biotechnology. 2013 Sep 1;31(9):822- 6.
- Palindromic Repeats CRISPR-associated IS. CRISPR-P: a web tool for synthetic singleguide RNA design of CRISPR-system in plants. Molecular plant. 2014 Sep;7:1494-6.
- Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature biotechnology. 2013 Sep 1;31(9):827- 32.
- Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013 Feb 28;152(5):1173-83.
- Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell. 2013 Dec 5;13(6):659-62.
- Charo RA, Greely HT. CRISPR critters and CRISPR cracks. The American Journal of Bioethics. 2015 Dec 2;15(12):11-7.
- Pennisi E. Gene drive turns mosquitoes into malaria fighters. Science. 2015 Nov 27;350(6264):1014-.
- Oye KA, Esvelt K, Appleton E, Catteruccia F, Church G, Kuiken T, Lightfoot SB, McNamara J, Smidler A, Collins JP. Regulating gene drives. Science. 2014 Aug 8;345(6197):626-8.
- Jasanoff S, Hurlbut JB, Saha K. CRISPR Democracy: Gene Editing and the Need for Inclusive Deliberation. Issues in Science and Technology. 2015 Oct 1;32(1):37.
- Fletcher J. Ethical aspects of genetic controls: Designed genetic changes in man. New England Journal of Medicine. 1971 Sep 30;285(14):776-83.
- Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & cell. 2015 May;6:363-72.
- Callaway E. UK scientists gain licence to edit genes in human embryos. Nature. 2016 Feb;530(7588):18-.
- Harris J. Germline manipulation and our future worlds. The American Journal of Bioethics. 2015 Dec 2;15(12):30-4.