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I will update section Precision of engineered nucleases to add efficiency of the different methods. I will add possible applications of CRISPR, mostly in the prospects and limitations section.

Precision and efficiency of engineered nucleases

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Meganucleases method of gene editing is the least efficient of the methods mentioned above. Due to the nature of its DNA-binding element and the cleaving element, it is limited to recognizing one potential target every 1,000 nucleotides.[1] ZFN was developed to overcome the limitations of meganuclease. The number of possible targets ZFN can recognized was increased to one in every 140 nucleotides.[1] However, both methods are unpredictable due to the ability of their DNA-binding elements affecting each other. As a result, high degrees of expertise and lengthy and costly validations processes are required.

TALE nucleases being the most precise and specific method yields a higher efficiency than the previous two methods. It achieves such efficiency because the DNA-binding element consists of an array of TALE subunits, each of them having the capability of recognizing a specific DNA nucleotide chain independent from others, resulting in a higher number of target sites with high precision. New TALE nucleases take about one week and a few hundred dollars to create, with specific expertise in molecular biology and protein engineering.[1]

CRISPR nucleases have a slightly lower precision when compared to the TALE nucleases. This is caused by the need of having a specific nucleotide at one end in order to produce the guide RNA that CRISPR uses to repair the double-strand break it induces. It has been shown to be the quickest and cheapest method, only costing less than two hundred dollars and a few days of time.[1] CRISPR also requires the least amount of expertise in molecular biology as the design lays in the guide RNA instead of the proteins. One major advantage that CRISPR has over the ZFN and TALEN methods is that it can directed to target different DNA sequences using its ~80nt CRISPR sgRNAs, while both ZFN and TALEN methods required construction and testing of the proteins created for targeting each DNA sequence.[2]

Because off-target activity of an active nuclease would have potentially dangerous consequences at the genetic and organismal levels, the precision of meganucleases, ZFNs, CRISPR, and TALEN-based fusions has been an active area of research. While variable figures have been reported, ZFNs tend to have more cytotoxicity than TALEN methods or RNA-guided nucleases, while TALEN and RNA-guided approaches tend to have the greatest efficiency and fewer off-target effects.[3] Based on the maximum theoretical distance between DNA binding and nuclease activity, TALEN approaches result in the greatest precision.[1]

 Applications

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As of 2012 efficient genome editing had been developed for a wide range of experimental systems ranging from plants to animals, often beyond clinical interest, and was becoming a standard experimental strategy in research labs.[4] The recent generation of rat, zebrafish, maize and tobacco ZFN-mediated mutants and the improvements in TALEN-based approaches testify to the significance of the methods, and the list is expanding rapidly. Genome editing with engineered nucleases will likely contribute to many fields of life sciences from studying gene functions in plants and animals to gene therapy in humans. For instance, the field of synthetic biology which aims to engineer cells and organisms to perform novel functions, is likely to benefit from the ability of engineered nuclease to add or remove genomic elements and therefore create complex systems.[4] In addition, gene functions can be studied using stem cells with engineered nucleases.

Listed below are some specific tasks this method can carry out:

Overview of GEEN workflow and editing possibilities

Targeted gene modification in plants

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Genome editing using meganucleases,[5] ZFNs, and TALEN provides a new strategy for genetic manipulation in plants and are likely to assist in the engineering of desired plant traits by modifying endogenous genes. For instance, site-specific gene addition in major crop species can be used for 'trait stacking' whereby several desired traits are physically linked to ensure their co-segregation during the breeding processes.[6] Progress in such cases have been recently reported in Arabidopsis thaliana[7][8][9] and Zea mays. In Arabidopsis thaliana, using ZFN-assisted gene targeting, two herbicide-resistant genes (tobacco acetolactate synthase SuRA and SuRB) were introduced to SuR loci with as high as 2% transformed cells with mutations.[10] In Zea mays, disruption of the target locus was achieved by ZFN-induced DSBs and the resulting NHEJ. ZFN was also used to drive herbicide-tolerance gene expression cassette (PAT) into the targeted endogenous locus IPK1 in this case.[11] Such genome modification observed in the regenerated plants has been shown to be inheritable and was transmitted to the next generation.[11]

In addition, TALEN-based genome engineering has been extensively tested and optimized for use in plants.[12] TALEN fusions have also been used to improve the quality of soybean oil products[13] and to increase the storage potential of potatoes[14]

Several optimizations need to be made in order to improve editing plant genomes using ZFN-mediated targeting.[15] These include the reliable design and subsequent test of the nucleases, the absence of toxicity of the nucleases, the appropriate choice of the plant tissue for targeting, the routes of introduction or induction of enzyme activity, the lack of off-target mutagenesis, and a reliable detection of mutated cases.[15]

Gene therapy

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The ideal gene therapy practice is that which replaces the defective gene with a normal allele at its natural location. This is advantageous over a virally delivered gene as there is no need to include the full coding sequences and regulatory sequences when only a small proportions of the gene needs to be altered as is often the case.[16] The expression of the partially replaced genes is also more consistent with normal cell biology than full genes that are carried by viral vectors.

Gene targeting through ZFNs or TALEN-based approaches can also be used to modify defective genes at their endogenous chromosomal locations. Examples include the treatment of X-linked severe combined immunodeficiency (X-SCID) by ex vivo gene correction with DNA carrying the interleukin-2 receptor common gamma chain (IL-2Rγ)[17] and the correction of Xeroderma pigmentosum mutations in vitro using TALEN.[18] Insertional mutagenesis by the retroviral vector genome induced leukemia in some patients, a problem that is predicted to be avoided by these technologies. However, ZFNs may also cause off-target mutations, in a different way from viral transductions. Currently many measures are taken to improve off-target detection and ensure safety before treatment.

In 2011, Sangamo BioSciences (SGMO) introduced the Delta 32 mutation (a suppressor of CCR5 gene which is a co-receptor for HIV-1 entry into T cells therefore enabling HIV infection) using Zinc Finger Nuclease (ZFN). Their results were presented at the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) held in Chicago from September 17–20, 2011.[19] Researchers at SGMO mutated CCR5 in CD4+ T cells and subsequently produced an HIV-resistant T-cell population.[20]

Gene editing is used to generate modified custom immune cells. For example, recent report indicated that T cells could be modified to inactivate the glucocorticoid receptor; the resulting immune cells are fully functional but resistant to the effects of commonly used corticosteroids.[21] Similarly, scientists at Cellectis recently generated custom T-cells expressing chimeric antigen receptors using TALEN technology.[22] These T-cells can be engineered to be resistant to anti-cancer drugs and to invoke immune responses against targets of interest.[23]

The first clinical use of TALEN-based genome editing was in the treatment of CD19+ acute lymphoblastic leukemia in an 11-month old child.[24] Modified donor T cells were engineered to attack the leukemia cells, to be resistant to Alemtuzumab, and to evade detection by the host immune system after introduction. A few weeks after therapy, the patient's condition improved. Though physicians were cautious, the patient was still in remission more than one year after treatment.[25][26][27]

Eradicating diseases

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CRISPR-Cas9 is being used in conjunction with gene drives as a potential to alter the traditional Mendelian laws of inheritance. Researchers have successfully used CRISPR-Cas9 gene drives to modify genes associated with sterility in A. gambiae, the vector for malaria.[28] If used widespread, this technique could suppress A. gambiae populations and with it malaria. Alternate genes are being researched that effect the vectors ability to carry the disease instead of causing intentional sterility and possible extinction. This technique has further implications in eradicating other vector borne diseases such as yellow fever, dengue, Zika, West Nile, Schistomiasis, Leishmaniasis and Lymes disease.

Given that there is currently no approved Lyme vaccine for humans, but there is for dogs, Kevin Esvelt, an associate professor of biological engineering at MIT has proposed using the vaccine on white-footed mice. If effective, CRISPR-Cas9 gene drives could be used in conjunction with genes associated with antibody forming in white-footed mice and then planted in the cells of mouse gametes. Esvelt is currently seeking approval of the residence of Nantucket, MA to utilize the island as a test environment for his experiment.[29]

The CRISPR-Cas9 system can be programmed to modulate the population any bacterial species by targeting clinical genotypes or epidemiological isolates. It can selectively enable the beneficial bacterial species over the harmful ones by eliminating pathogen, which gives it an advantage over broad-spectrum antibiotics.[2]

Antiviral applications for therapies targeting human viruses such as HIV, herpes, and hepatitis B virus are under research. CRISPR can be used to target the virus or the host to disrupt genes encoding the virus cell-surface receptor proteins. [2]

Extensive research is being done on CRISPR-Cas9 in correcting genetic mutations which cause genetic diseases such as Down syndrome, spina bifida, anencephaly, and Tuner and Klinefelter syndromes using targeted gene therapy, if these genetic mutations are identified early enough in the embryo stages, which we currently already have the capability to do so consistently. With many of the genetic diseases caused from one overexpressed gene, CRISPR-Cas9 can be used to silence an entire chromosome, or delete the overexpressed gene with Cas9 endonuclease cutting.[30]

Other studies are being done on using CRISPR to target specific genes in cancer cells in hopes of using genome editing to inhibit cell proliferation and tumorigenicity of cancer cells.[31]

 Prospects and limitations

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In the future, an important goal of research into genome editing with engineered nucleases must be the improvement of the safety and specificity of the nucleases. For example, improving the ability to detect off-target events can improve our ability to learn about ways of preventing them. In addition, zinc-fingers used in ZFNs are seldom completely specific, and some may cause a toxic reaction. However, the toxicity has been reported to be reduced by modifications done on the cleavage domain of the ZFN.[32]

In addition, research by Dana Carroll into modifying the genome with engineered nucleases has shown the need for better understanding of the basic recombination and repair machinery of DNA. In the future, a possible method to identify secondary targets would be to capture broken ends from cells expressing the ZFNs and to sequence the flanking DNA using high-throughput sequencing.[32]

Because of the ease of use and cost-efficiency of CRISPR, extensive research is currently being done on it. There are now more publications on CRISPR than ZFN and TALEN despite how recent the discovery of CRISPR is.[2] Both CRISPR and TALEN are favored to be the choices to be implemented in large-scale productions due to their precision and efficiency.

Genome editing occurs also as a natural process without artificial genetic engineering. The agents that are competent to edit genetic codes are viruses or subviral RNA-agents.[33]

Although GEEN has higher efficiency than many other methods in reverse genetics, it is still not highly efficient; in many cases less than half of the treated populations obtain the desired changes.[34] For example, when one is planning to use the cell's NHEJ to create a mutation, the cell's HDR systems will also be at work correcting the DSB with lower mutational rates.

Traditionally, mice have been the most common choice for researchers as a host of a disease model. CRISPR can help bridge the gap between this model and human clinical trials by creating transgenic disease models in larger animals such as pigs, dogs, and non-human primates. [35][36] Using the CRISPR-Cas9 system, the programmed Cas9 protein and the sgRNA can be directly introduced into fertilized zygotes to achieve the desired gene modifications when creating transgenic models in rodents. This allows bypassing of the usual cell targeting stage in generating transgenic lines, and as a result, it reduces generation time by 90%. [36]

One potential that CRISPR brings with its effectiveness is the application of xenotransplantation. In previous research trials, CRISPR demostrated the ability to target and eliminate endogenous retroviruses, which reduces the risk of trasmitting diseases and reduces immune barriers.[2] Eliminating these problems improves donor organ function, which brings this application closer to a reality.

Eradication of diseases

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Some researches see potential to end diseases that have caused many deaths and suffering in the past. Similarly to the idea of designer babies, genome editing could potentially correct genetic mutations which cause genetic diseases such as Down syndrome, spina bifida, anencephaly, and Tuner and Klinefelter syndromes, if these genetic mutations are identified early enough in the embryo stages, which we currently already have the capability to do so consistently.

Genome editing on mosquitoes could stop them from carrying malaria and other mosquito-borne diseases that are deadly to humans.

Other studies are being done on using CRISPR to target specific genes in cancer cells in hopes of using genome editing to inhibit cell proliferation and tumorigenicity of cancer cells.

Human enhancement

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Many transhumanists see genome editing as a potential tool for human enhancement.[37][38][39] Australian biologist and Professor of Genetics David Andrew Sinclair notes that "the new technologies with genome editing will allow it to be used on individuals [...] to have [...] healthier children" - designer babies.[40] According to a September 2016 report by the Nuffield Council on Bioethics in the future it may be possible to enhance people with genes from other organisms or wholly synthetic genes to for example improve night vision and sense of smell.[41][42]

The American National Academy of Sciences and National Academy of Medicine issued a report in February 2017 giving qualified support to human genome editing.[43] They recommended that clinical trials for genome editing might one day be permitted once answers have been found to safety and efficiency problems "but only for serious conditions under stringent oversight."[44]

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  7. ^ Townsend, J.A. et al., High-frequency modification of plant genes using engineered zinc-finger nucleases" Nature 459 (7245), 442-445 (2009).
  8. ^ Zhang, F. et al., High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases" Proceedings of the National Academy of Sciences 107 (26), 12028-12033 (2009).
  9. ^ Osakabe, K., Osakabe, Y., & Toki, S., Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases" Proceedings of the National Academy of Sciences 107 (26), 12034-12039 (2010).
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  12. ^ Zhang, Yong; Zhang, Feng; Li, Xiaohong; Baller, Joshua A.; Qi, Yiping; Starker, Colby G.; Bogdanove, Adam J.; Voytas, Daniel F. (2013-01-01). "Transcription activator-like effector nucleases enable efficient plant genome engineering". Plant Physiology. 161 (1): 20–27. doi:10.1104/pp.112.205179. ISSN 1532-2548. PMC 3532252. PMID 23124327.
  13. ^ Haun, William; Coffman, Andrew; Clasen, Benjamin M.; Demorest, Zachary L.; Lowy, Anita; Ray, Erin; Retterath, Adam; Stoddard, Thomas; Juillerat, Alexandre (2014-09-01). "Improved soybean oil quality by targeted mutagenesis of the fatty acid desaturase 2 gene family". Plant Biotechnology Journal. 12 (7): 934–940. doi:10.1111/pbi.12201. ISSN 1467-7652. PMID 24851712.
  14. ^ Clasen, Benjamin M.; Stoddard, Thomas J.; Luo, Song; Demorest, Zachary L.; Li, Jin; Cedrone, Frederic; Tibebu, Redeat; Davison, Shawn; Ray, Erin E. (2015-04-07). "Improving cold storage and processing traits in potato through targeted gene knockout". Plant Biotechnology Journal. 14 (1): 169–76. doi:10.1111/pbi.12370. ISSN 1467-7652. PMID 25846201.
  15. ^ a b Puchta, H. & Hohn, B., Breaking news: Plants mutate right on target" Proceedings of the National Academy of Sciences 107 (26), 11657-11658 (2010).
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  18. ^ Dupuy, Aurélie; Valton, Julien; Leduc, Sophie; Armier, Jacques; Galetto, Roman; Gouble, Agnès; Lebuhotel, Céline; Stary, Anne; Pâques, Frédéric (2013-11-13). "Targeted Gene Therapy of Xeroderma Pigmentosum Cells Using Meganuclease and TALEN™". PLOS ONE. 8 (11): e78678. doi:10.1371/journal.pone.0078678. PMC 3827243. PMID 24236034.
  19. ^ Sangamo BioSciences Announces Presentation of Groundbreaking Clinical Data From ZFN Therapeutic for HIV/AIDS at ICAAC 2011 (2011).
  20. ^ Perez, E.E. et al., Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotech 26 (7), 808-816 (2008).
  21. ^ Menger, Laurie; Gouble, Agnes; Marzolini, Maria A. V.; Pachnio, Annette; Bergerhoff, Katharina; Henry, Jake Y.; Smith, Julianne; Pule, Martin; Moss, Paul (2015-01-01). "TALEN-mediated genetic inactivation of the glucocorticoid receptor in cytomegalovirus-specific T cells". Blood. 126 (26): blood-2015-08-664755. doi:10.1182/blood-2015-08-664755. ISSN 0006-4971. PMID 26508783.
  22. ^ Valton, Julien; Guyot, Valérie; Marechal, Alan; Filhol, Jean-Marie; Juillerat, Alexandre; Duclert, Aymeric; Duchateau, Philippe; Poirot, Laurent (2015-09-01). "A Multidrug-resistant Engineered CAR T Cell for Allogeneic Combination Immunotherapy". Molecular Therapy. 23 (9): 1507–1518. doi:10.1038/mt.2015.104. ISSN 1525-0024. PMC 4817890. PMID 26061646.
  23. ^ Poirot, Laurent; Philip, Brian; Schiffer-Mannioui, Cécile; Clerre, Diane Le; Chion-Sotinel, Isabelle; Derniame, Sophie; Bas, Cécile; Potrel, Pierrick; Lemaire, Laetitia (2015-07-16). "Multiplex genome edited T-cell manufacturing platform for "off-the-shelf" adoptive T-cell immunotherapies". Cancer Research. 75 (18): 3853–3864. doi:10.1158/0008-5472.CAN-14-3321. ISSN 0008-5472. PMID 26183927.
  24. ^ Pollack, Andrew (2015-11-05). "A Cell Therapy Untested in Humans Saves a Baby With Cancer". The New York Times. ISSN 0362-4331. Retrieved 2015-11-30.
  25. ^ Henry, Robin (2017-02-19). "Leukaemia cure hopes rise as girl is gene‑edited". The Times. Retrieved 2017-02-27. {{cite news}}: Unknown parameter |subscription= ignored (|url-access= suggested) (help)
  26. ^ "Paper: First Clinical Application of Talen Engineered Universal CAR19 T Cells in B-ALL". ash.confex.com. Archived from the original on 2016-02-05. Retrieved 2015-11-30.
  27. ^ "Science Magazine: Baby's leukemia recedes after novel cell therapy". Retrieved 2015-11-30.
  28. ^ Hammond, Andrew; Galizi, Roberto; Kyrou, Kyros; Simoni, Alekos; Siniscalchi, Carla; Katsanos, Dimitris; Gribble, Matthew; Baker, Dean; Marois, Eric (2016). "A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae". Nature Biotechnology. 34 (1): 78–83. doi:10.1038/nbt.3439. PMC 4913862. PMID 26641531.
  29. ^ Specter, M. (2017). Rewriting the Code of Life. The New Yorker, (43).
  30. ^ Mentis, A. F. (2016-12-01). "Epigenomic engineering for Down syndrome". Neuroscience and Biobehavioral Reviews. 71: 323–327. doi:10.1016/j.neubiorev.2016.09.012. ISSN 1873-7528. PMID 27646312. S2CID 24192441.
  31. ^ Wang, Huajing; Sun, William (2017-01-28). "CRISPR-mediated targeting of HER2 inhibits cell proliferation through a dominant negative mutation". Cancer Letters. 385: 137–143. doi:10.1016/j.canlet.2016.10.033. ISSN 1872-7980. PMID 27815036.
  32. ^ a b Carroll, D., Progress and prospects: Zinc-finger nucleases as gene therapy agents. Gene Ther 15 (22), 1463-1468 (2008).
  33. ^ Witzany, G (2011). "The agents of natural genome editing". J Mol Cell Biol. 3 (3): 181–189. doi:10.1093/jmcb/mjr005. PMID 21459884.
  34. ^ Townsend, J.A. et al., High-frequency modification of plant genes using engineered zinc-finger nucleases" Nature 459 (7245), 442-445 (2009).
  35. ^ Im, Wooseok; Moon, Jangsup; Kim, Manho (2017-04-11). "Applications of CRISPR/Cas9 for Gene Editing in Hereditary Movement Disorders". Journal of Movement Disorders. 9 (3): 136–143. doi:10.14802/jmd.16029. ISSN 2005-940X. PMC 5035944. PMID 27667185.
  36. ^ a b Hsu, Patrick D.; Lander, Eric S.; Zhang, Feng (2014-06-05). "Development and Applications of CRISPR-Cas9 for Genome Engineering". Cell. 157 (6): 1262–1278. doi:10.1016/j.cell.2014.05.010. ISSN 0092-8674. PMC 4343198. PMID 24906146.
  37. ^ Pearlman, Alex. "Geneticists Are Concerned Transhumanists Will Use CRISPR on Themselves". Vice Motherboard. Retrieved 26 December 2016.
  38. ^ Jorgensen, Ellen. "How DIY bio-hackers are changing the conversation around genetic engineering". The Washington Post. Retrieved 26 December 2016.
  39. ^ "Human Enhancement". Pew Research Center. Retrieved 26 December 2016.
  40. ^ Regalado, Antonio. "Engineering the Perfect Baby". MIT Technology Review. Retrieved 26 December 2016.
  41. ^ Sample, Ian (30 September 2016). "Experts warn home 'gene editing' kits pose risk to society". The Guardian. Retrieved 26 December 2016.
  42. ^ "Genome editing: an ethical review" (PDF). Nuffield Council on Bioethics. September 2016. Retrieved 27 December 2016.
  43. ^ Harmon, Amy (2017-02-14). "Human Gene Editing Receives Science Panel's Support". The New York Times. ISSN 0362-4331. Retrieved 2017-02-17.
  44. ^ "Scientists OK genetically engineering babies". New York Post. Reuters. 2017-02-14. Retrieved 2017-02-17.
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