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This is an old revision of this page, as edited by Jez.chow (talk | contribs) at 23:00, 15 March 2017 (Added more potential uses in genome editing). The present address (URL) is a permanent link to this revision, which may differ significantly from the current revision.

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

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. 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. 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.

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. CRISPR also requires the least amount of expertise in molecular biology as the design lays in the guide RNA instead of the proteins.

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.[1] Based on the maximum theoretical distance between DNA binding and nuclease activity, TALEN approaches result in the greatest precision.[2]

 Prospects and limitations

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.[3]

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.[3]

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.

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.[4]

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.[5] 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.

Human enhancement

Many transhumanists see genome editing as a potential tool for human enhancement.[6][7][8] 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.[9] 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.[10][11] However, correcting genetic mutations are more likely to be the first genetic edits. This could potentially eradicate 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.

The American National Academy of Sciences and National Academy of Medicine issued a report in February 2017 giving qualified support to human genome editing.[12] 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."[13]

  1. ^ Kim, Hyongbum; Kim, Jin-Soo (2014-04-02). "A guide to genome engineering with programmable nucleases". Nature Reviews Genetics. 15 (5): 321–334. doi:10.1038/nrg3686.
  2. ^ Boglioli, Elsy; Richard, Magali. "Rewriting the book of life: a new era in precision genome editing" (PDF). Boston Consulting Group. Retrieved November 30, 2015.
  3. ^ a b Carroll, D., Progress and prospects: Zinc-finger nucleases as gene therapy agents. Gene Ther 15 (22), 1463-1468 (2008).
  4. ^ Witzany, G (2011). "The agents of natural genome editing". J Mol Cell Biol. 3 (3): 181–189. doi:10.1093/jmcb/mjr005. PMID 21459884.
  5. ^ Townsend, J.A. et al., High-frequency modification of plant genes using engineered zinc-finger nucleases" Nature 459 (7245), 442-445 (2009).
  6. ^ Pearlman, Alex. "Geneticists Are Concerned Transhumanists Will Use CRISPR on Themselves". Vice Motherboard. Retrieved 26 December 2016.
  7. ^ Jorgensen, Ellen. "How DIY bio-hackers are changing the conversation around genetic engineering". The Washington Post. Retrieved 26 December 2016.
  8. ^ "Human Enhancement". Pew Research Center. Retrieved 26 December 2016.
  9. ^ Regalado, Antonio. "Engineering the Perfect Baby". MIT Technology Review. Retrieved 26 December 2016.
  10. ^ Sample, Ian (30 September 2016). "Experts warn home 'gene editing' kits pose risk to society". The Guardian. Retrieved 26 December 2016.
  11. ^ "Genome editing: an ethical review" (PDF). Nuffield Council on Bioethics. September 2016. Retrieved 27 December 2016.
  12. ^ Harmon, Amy (2017-02-14). "Human Gene Editing Receives Science Panel's Support". The New York Times. ISSN 0362-4331. Retrieved 2017-02-17.
  13. ^ "Scientists OK genetically engineering babies". New York Post. Reuters. 2017-02-14. Retrieved 2017-02-17. {{cite web}}: Cite has empty unknown parameter: |dead-url= (help)
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