Few scientific breakthroughs have been as important as the discovery of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) technique as a gene editing tool. CRISPR/Cas9 is a natural system that provides bacteria with an adaptive response against viruses.  In 2012, Jennifer Doudna and Emmanuelle Charpentier published a study in which they detailed how this system could be used to perform programmed gene editing in different cell types.  Other gene editing techniques had previously been discovered, such as TALENs (transcription activator-like effector nucleases) or ZFNs (zinc finger nucleases). However, their complexity of use, high cost and poor or moderate efficacy has prevented their widespread application, even though good results had been obtained in some cases. The CRISPR/Cas9 technique overcomes these three obstacles, so it has spread very quickly to laboratories around the world, relegating the former techniques to second place. Thus, the number of publications in this field is increasing rapidly. It seems fair to say that, with the discovery of CRISPR/Cas9, gene editing is here to stay.

History

In 1987, the first article was published that described repeated sequences in the genome of bacteria, specifically Escherichia coli (2). It was initially considered that these repeated sequences lacked any function.

In 1993, Spaniard Francisco Martínez Mojica described that same sequence in another type of bacteria, Haloferax mediterranei, whose habitat is found only and exclusively in the salt flats of Santa Pola on the shores of the Mediterranean in Spain (3). In 2000, Martínez Mojica described this same sequence in another group of bacteria, and called them short regularly spaced repeats (SRSRs) (4). Two years later, Ruud Jansen identified some genes associated with these repeat sequences and, with Martínez Mojica’s consent, renamed the repeat sequences, which came to be called clustered regularly interspaced short palindromic repeats (CRISPR) (5). In 2005, Martínez Mojica identified similarities between the spacers associated with CRISPR described by Ruud Jansen and the genetic material of certain viruses that affect bacteria. It was then that the CRISPR system was identified as a bacterial system of defence against viruses, a system than can be passed on to successive generations of bacteria (6). Martínez Mojica’s discoveries laid the foundation for the subsequent development of the gene editing technique (see HERE), which earned him a nomination for the 2016 Nobel Prize for Medicine (although he did not win).

In 2012, the team led by Doudna and Charpentier made the first “cut” using the CRISPR/Cas9 system in a test tube, and suspected that the same could be done in other types of cells, such as eukaryotic cells, and that it could be used for gene editing. Both investigators were awarded the Princess of Asturias prize for scientific and technical research in 2015 for their work .

Later that same year, Feng Zhang and his team managed to make the first cut using CRISPR/Cas9 on the genome of a live mammalian cell (7). Zhang inscribed this finding in the United States patent register. This register is currently in a legal dispute with researchers Doudna and Charpentier.

How CRISPR/Cas works

Bacteria are prokaryotic cells, which means that their DNA is not protected within a cell nucleus, but is “loose” in the cytoplasm. This leads to the need for a defence system, the CRISPR/Cas system, which has been shown to be very important for bacterial survival, because if the CRISPR sequences are removed, the bacteria die (6).

In the natural environment, when a bacterium detects the entry of a viral DNA, it sends an RNA sequence that can “copy” up to 20 nucleotides of the virus DNA. The copy sequence then binds to the Cas9 cutting protein. Once bound in a unique complex, the RNA with the 20 copied nucleotides locates the binding site in the viral DNA, binds, and the Cas9 protein makes a cut in the invader’s DNA.

Use of gene editing techniques in medicine, environment, agriculture and livestock applications.

We are only beginning to glimpse the enormous possibilities offered by this new biotechnology tool, which as well as a multitude of applications in the medical field, has environmental, agricultural and livestock applications. The ethical aspects of these applications are discussed in our Observatory.

The healthcare applications arouse most interest due to their direct impact on people’s lives, and at the same time the most controversy, mainly in relation to germline genetic modification (gametes and embryos).

Hundreds of studies on this aspect  are being conducted, with multiple and diverse objectives, from the design of new methods to combat difficult-to-treat diseases, such as HIV and several types of cancer, to the possibility of treating genetic diseases. In this sense, what would be the first trial in humans in the United States was recently approved . Researchers in this trial will select 18 subjects with different types of melanoma, myeloma and carcinoma who do not respond to standard treatments. The expectations placed on this trial are huge, as it raises the possibility of combating cancer in a way that is efficient and relatively non-invasive, dispensing with current treatments based on surgery, chemotherapy and radiotherapy (8). However, Chinese scientists have raced ahead, and have used the technique in a patient with lung cancer, who will be monitored to check the safety and efficacy of the method.

Despite considerable reservations among scientists (9) (10), studies have already been conducted on non-viable embryos (11) (12), both in China .

Gene editing germline a bioethics assessement

Gene editing germline. Great possibilities is offered by this new biotechnology tool but it involves objective ethical problems when affects human germlineWhile it is true that the scientific community almost unanimously agrees that gene editing for non-therapeutic use, e.g. to select eye colour — in other words, for genetic “enhancement” — is objectionable because it is ethically unacceptable, it is also true that the community is currently divided into two large groups: those who reject this technology because of the ethical problems involved in germline therapy, and those who believe that it can have a legitimate use to prevent genetic diseases. This division only refers to the use of gene editing on the germline, as there is a wide consensus in favour of its use in somatic cells.

Nevertheless, it seems that the tendency is to accept research on embryos but prevent their implantation in the uterus (see HERE). In fact, studies of this type have already been authorised in England (see HERE). From our point of view, this is morally unacceptable, as the human embryo has equal dignity to a person already born. You can read our reflection on genetic modification of human embryos to treat diseases HERE.

A study conducted on 39 countries to try to clarify the current legal situation with respect to germline gene editing (13) revealed the following:

  • 25 countries prohibit it by law. This group includes Australia, Austria, Belgium, Brazil, Bulgaria, Canada, Costa Rica, Denmark, Finland, France, Germany, Israel, Italy, Lithuania, Mexico, New Zealand, Singapore, South Korea, Sweden, Switzerland, Czech Republic, Norway, United Kingdom, Portugal and Spain. This is the largest group, and encompasses most neighbouring countries.
  • 4 prohibit it using directives, which are less restrictive than a law, and can be subject to modifications more easily than a law. This is the case of China, Japan, India and Ireland.
  • 9 are ambiguous in their laws. This group includes Argentina, Chile, Colombia, Greece, Iceland, Peru, Russia, Slovakia and South Africa.
  • It is restrictive. This is the interesting case of the United States. There are 2 state agencies involved. The FDA (Food and Drug Administration) regulate clinical trials, while the NIH (National Institutes of Health) restrict their practical application in humans.

Gene editing germline modification in humans

The panorama of current international regulation in this respect suggests that human germline genetic modification is not completely banned, as there are areas for research in countries classified as “ambiguous” in their regulation. In the case of China and the United Kingdom, studies have been authorised in this respect, despite their regulations. Likewise, the four countries named previously with bans through directives could lift these bans when the safety of germline gene correction improves.

A report from the Nuffield Council on Bioethics  on gene editing, published in September 2016 (14), notes that, in addition to reproductive medicine, another vitally important area to address is the application of these techniques in agriculture.

There is also the possibility of modifying ecosystems. This is the case of mosquitoes, major vectors that transmit many difficult-to-control diseases. Scientists have speculated about the possibility of genetically altering a series of individuals and releasing the modified insects into the natural environment, so that they interact with their respective populations and thus spread the desired genetic modification. In this respect, it has been proposed that CRISPR be combined with gene drive (see HERE), which would allow almost any gene in any species with sexual reproduction to be altered, and to spread the alterations produced through wild populations. Nevertheless, the consequences of modifying an entire population are unknown and worrisome . Hence, the United States National Academy of Sciences launched guidelines for responsible conduct of gene drive-related research.

In order to discuss the scientific, medical, legal and ethical implications of these advances, Doudna convened a meeting with specialists in various scientific fields in January 2015 in Napa (California, US), identifying a series of steps to be followed  (9).

Bioethical assessment

In the case of use of gene editing in the primary industry, agriculture and livestock, we cannot but be in favour of all those uses that involve a benefit for humanity. A case-by-case analysis would be necessary, discarding those uses in which the benefit sought is lower than the potential risk of the genetic manipulation. For example, manipulation of plant species so that they are resistant to various types of plagues presents a large benefit for mankind, namely by avoiding the use of pesticides and other chemical products to protect crops, as these can be dispersed through the air, reaching populated areas or filtering through to subterranean aquifers. It should be noted that, sometimes, the problem may not be a safety concern, but rather a question of justice. It must be guaranteed that any advance in this field will not lead to a form of exploitation. Moreover, information to the final consumer should be regulated by labelling the origin of the product.

We must be more cautious, if possible, with interventions on ecosystems, as any alteration could lead to incalculable, extremely serious problems, even on a world scale, since the natural environment does not respect political boundaries.

As regards medical applications, the use of gene editing techniques on somatic cells is likely to occur sooner than other applications, and in fact, the first study of this type has already been authorised. Although hugely promising, studies in this field still have a long way to go. Scientists have great expectations in this respect, and a multitude of studies are being conducted, with enormous investments of resources. In order for this technique to be acceptable, its safety must be improved, and it should be used only in those diseases for which there is currently no effective treatment, or in diseases in which current treatments involve major side effects; moreover, their success rate and possibility of side effects should be the same as those of current treatments.

However, if there is one use of this technique that generates more controversy, it is its application in germ cells, above all because of the hereditary nature of the changes made in the DNA of these cells. The history of genetics is relatively recent, and there are still many gaps in our knowledge. As this has to do with the germline and inheritable modifications, we must harbour no doubts about any mechanism involved. If gene editing of somatic cells requires safety and efficacy as good as, or better than, those of traditional medicines or vaccines, then in germline modification these must be 100%.

In addition, application of this technique requires in vitro fertilisation, with the ethical difficulties that this entails. Moreover, their use already includes preimplantation genetic diagnosis, which implies that gene editing in this case might not be useful.

Although the potential benefits are many, the number of people that might benefit is small, and the risks to assume are incalculable, irreversibly affecting the entire species. Germline gene editing is ethically unacceptable.

Limitations of CRISPR-Cas9 and alternatives

Despite the great potential of the CRISPR-Cas9 technique, it also has its limitations, for which several alternatives have been proposed (15).

The components of the CRISPR-Cas9 system — an enzyme called Cas9 and a strand of RNA that directs this enzyme to the desired sequence — are too large to be introduced into the genome of the virus most commonly used in gene therapy to transport foreign genetic material into human cells. One solution presents in the form of a mini-Cas9, which was obtained from the bacterium Staphylococcus aureus (16). This enzyme is small enough to fit inside the virus. Last December, two groups used the mini-Cas9 in mice to correct the gene responsible for Duchenne muscular dystrophy (17) (18).

Cas9 will not always cut where intended – a certain DNA sequence must be nearby for that to happen. This demand is easily met in many genomes, but can be a limitation in some experiments. Researchers are looking at microbes to obtain enzymes with different sequence requirements so that they can expand the number of sequences that can be modified. One of these enzymes, called Cpf1, could become an attractive alternative. Smaller than Cas9, it has different sequence requirements and is highly specific (19) (20). Another enzyme, called C2c2, targets RNA instead of DNA, a characteristic that has great potential for studying RNA and fighting against viruses with RNA genomes (21).

Many laboratories use CRISPR-Cas9 only to delete sections in a gene, thereby suppressing its function. Those who want to exchange one sequence for another face a more difficult task. When Cas9 cuts the DNA, the cell often makes mistakes as it joins the loose ends together, thereby obtaining the desired deletions. However, researchers who want to rewrite a DNA sequence depend on a different repair pathway that can insert a new sequence – a process that occurs at a much lower frequency. Recently, though, researchers announced that they had disabled Cas9 and tied it to an enzyme that converts one DNA letter to another. The disabled Cas9 still targeted the sequence dictated by its guide RNA, but did not cut; instead, the attached enzyme exchanged the DNA letters, ultimately producing a T where once there was a C (22). A paper published recently in Science reported similar results (23).

In May, a paper in Nature Biotechnology (24) revealed a new gene-editing system. Researchers claimed that they could use a protein called NgAgo to cut the DNA at a predetermined site without needing a guide RNA or a specific neighbouring genome sequence. Instead, the protein — of bacterial origin — is programmed using a short DNA sequence that corresponds to the target area. Laboratories have nonetheless failed to reproduce the results so far, so the efficacy of this technique cannot be confirmed. Even so, there is still hope that proteins from the family to which NgAgo belongs ( Ago or Argonautes) made by other bacteria could work.

There are also other gene editing systems, some of which have existed for years. George Church, a geneticist at Harvard Medical School (Boston, US) did not use CRISPR for a large project that aimed to modify genes in bacteria. Instead, the team relied to a large degree on a system called lambda Red, which can be programmed to alter DNA sequences without the need for a guide RNA. Despite 13 years of study in Church’s laboratory, however, lambda Red works only in bacteria.

G. Church and Feng Zhang, a bioengineer at the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, say that their laboratories are also working on developing enzymes called integrases and recombinases for use as gene editors.

 

References

  1. Doudna J, Charpentier E, Jinek M, Chylinski K, Fonfara I, Hauer M. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. 2012 August 17; 337(6096): p. 816-821.
  2. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology. 1987 December; 169(12).
  3. Matinez Mojica FJ, Juez G, Rodriguez-Valera F. Transcription at different salinities of Haloferaxmediterranei sequences adjacent to partially modified PstI sites. Molecular microbiology. 1993 August; 9(3).
  4. Martinez Mojica FJ, Diez-Villaseñor C, Soria E, Juez G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bateria and mitochondria. Molecular microbiology. 2000 April; 36(1).
  5. Jansen R, van Embden JDA, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Molecular microbiology. 2002 March; 43(6).
  6. Martinez Mojica F, Diez-Villaseñor C, Garcia-Martinez J, Soria E. Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements. Molecular Evolution. 2005 February; 60(2).
  7. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. 2013 February 15; 339(6121): p. 819-823.
  8. Kaiser J. First proposed human test of CRISPR passes initial safety review. 2016. Available at: http://www.sciencemag.org/news/2016/06/human-crispr-trial-proposed.
  9. Baltimore, D., et al. A prudent path forward for genomic engineering and germline gene modification. Science. 2015. 348, 36-38.
  10. Lanphier, E., Urnov, F., Haecker, S. E., Werner, M. &Smolenski, J. Don’t edit the human germ line. Nature. 2015. 519, 410-411.
  11. Liang, P. et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015. 6, 363-372.
  12. Kang, X., et al. Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing. J Assist Reprod Genet. 2016 May;33(5):581-8. doi: 10.1007/s10815-016-0710-8. Epub 2016 Apr 6.
  13. Araki M, Ishii T. International regulatory landscape and integration of corrective genome editing into in vitro fertilization. Reproductive biology and endocrinology. 2014 November; 12(108).
  14. http://nuffieldbioethics.org/wp-content/uploads/Genome-editing-an-ethical-review.pdf
  15. Ledford, H. Beyond CRISPR: A guide to the many other ways to edit a genome. Nature. 2016 Aug 8;536(7615):136-7. doi: 10.1038/536136b.
  16. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015. 520, 186–191.
  17. Nelson, C. E. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016. 351, 403–407.
  18. Tabebordbar, M. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2016. 351, 407–411.
  19. Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nature Biotechnol. 2016. doi: 10.1038/nbt.3609.

Lucía Gómez – Tatay

Bioethics Observatory