Dr Heli Matilainen explores the lightning-fast development of targeted genome engineering.
We have rapidly entered the era of ‘next generation’ genetic engineering (GE). The revolution in this field is largely due to the development and introduction of targeted genome engineering/modification/editing techniques.
These new techniques have been widely adopted in all relevant scientific areas at a very fast pace. Recent major breakthroughs and developments have enabled genome editing/engineering to get to the ‘science fiction’ level.
These techniques promise fast, tailor-made modifications to the specific site of a genome (Kim and Kim 2014). This is in sharp contrast with the earlier genetically modified organisms (GMOs), which were all created by random integration of the foreign transgene to the genome of the host organism (Puchta and Fauser 2013) – in other words, when inserting genetic material, scientists cannot control where in the genome it will end up.
How does it work?
The new techniques also (for the most part) introduce engineered or synthetic genetic material to the cell as part of the process, but it is not necessarily added to the genome of the end product on purpose.
Targeted genome engineering employs programmable nucleases (DNA-cleaving proteins), which have been modified or altered – or guided by other means – to cut DNA from a specific point of the genome. The benefit of these programmable nucleases arises from the ability to ‘cut’ the DNA from the specific location, thus enabling targeted changes to be made. The cell’s internal DNA repair systems are employed to finish off the job, either joining the introduced foreign DNA material to the specific cut site, or by repairing the damaged DNA site (Fichtner et al. 2014, Kim and Kim 2014).
Programmable nucleases that have been developed include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases: CRISPR/Cas9 (Kim and Kim 2014). New applications, as well as improvements to the existing ones, are being developed and published as you read this.
What are the techniques used for?
The most powerful of the techniques developed so far, due to its ease of use and adaptability, is based on RNA-guided engineered nucleases, and is called CRISPR/Cas9 (CRISPR = clustered regularly interspaced short palindromic repeat) (Cong et al., 2013). Since its development in 2013, all areas of science it can be applied to have rapidly moved forward. (Ledford, 2016).
There are essentially no limits to what can be achieved. The new genome-editing techniques, especially CRISPR/Cas9, can be used to induce virtually any kind of changes in the genome of the target organism: adding new genes or shorter DNA sequences, deleting sequences or entire genes, introducing designed mutations and so on (Fichtner et al., 2014). New applications include silencing (turning off) and activating genes and inducing epigenetic changes in the genome in order to control expression (activity) of larger sets of genes in the genome (Ledford, 2016).
The versatility and huge potential of targeted genome editing using engineered nucleases is crystallised by Kim and Kim (2014): ‘With engineered nucleases, molecular breeders can now modify animal and plant genomes in a targeted manner to improve or alter essentially any trait at will’.
Traits that have been altered include for example enhancing the aromatic qualities of rice (Shan et al. 2015), or preventing browning of apples (Nishitani et al. 2016). Altering or improving pest resistance has been a major focus, an example being a wheat line with mutations in the mildew resistance locus (Wang et al. 2015). Dairy cattle can be engineered to produce some therapeutic agents to the milk (Jeong al 2016).
Successfully edited plants include crop species such as rice, soybean, tomato, wheat, tobacco, maize, potato, sweet orange, lettuce, barley (reviewed in Hilscher et al. 2016) and apple (Nishitani et al. 2016). Gene-edited farm animals created include goat, pig, rabbit, dairy cattle, and sheep (Reviewed in Lotti et al., 2017).
Targeted genome editing (CRISPR/Cas9) has already been used for human embryos to induce precise mutations to ‘establish principles for the introduction of precise genetic modifications in early human embryos’ (Kang et al. 2016). Targeted genome editing has also been applied to human-induced pluripotent stem cells, one of the aims being to use these genetically modified stem cells in cell therapy (reviewed in Brookhouser et al. 2017).
What are the risks?
There is still a great deal we don’t yet understand about the methods and the potential risks associated with these new gene-editing techniques. Companies developing the new techniques emphasise the ‘safety and accuracy’ of these techniques, however there are several serious safety concerns.
The so-called ‘off-target events’ where the genome is being cut and modified from a non-target site may pose unknown risks. All the major engineered nuclease-based techniques may cause off-target events, and there have been reports outlining substantial off-target events in more complex genomes, such as human (review in Kim and Kim, 2014).
Unknown risks are involved in the introduction of foreign material (DNA/RNA/engineered molecules) to the cells, plants or animals, as well as the exact effects of the changes (intended and off-target e.g. accidental) that are made to the genome. The genomes of living creatures are very complex, we have yet much to learn regarding their ingenious design and function.
It is thus impossible to predict the full impacts of the various GE products that are being engineered using gene-editing techniques.
As the crops (and animals) being developed are very diverse and have different traits, it can be expected that the potential adverse effects on human beings and on the environment will differ, and therefore case-by-case safety and risk analysis will be required (Eckerstorfer et al., 2014).
Importantly, many products created using these techniques cannot be detected using laboratory testing – unless the companies generating them add identifiers or similar tags to DNA.
How are they regulated around the world?
The fast pace of the development of targeted genome engineering has left regulatory bodies scratching their heads. Hilsher and colleagues (2017) summarise it: ‘The arrival of the genome editing technology and particularly its rapid penetration of commercial translational research has caught the regulatory authorities off-guard and the regulatory status of crop varieties developed with this technology needs to be clarified urgently.’
In the US, where GE regulation is based on product rather than the process, many genome-edited products do not fall within the scope of GMOs, and therefore do not need to be regulated as such (Hilscher et al., 2017 and Walz, 2016).
In the EU, GMOs are assessed according to process-based criteria, and this is causing problems because there may not be a way to identify a product that has been edited or modified using targeted gene modification. The report by the New Techniques Working Group, which was set up by EU regulatory authorities in 2007, was never officially published, leaving the regulatory status of new edited organisms uncertain (Fladung, 2016).
In New Zealand, the new genome engineering techniques remain regulated as GMOs under New Zealand law, for now (see www.beehive.govt.nz/release/gmo-regulations-clarified).
A threat to NZ’s GE-free status and organics
The rapid adoption of the new gene editing techniques poses a real threat to organics – and GE-free status of our food. The lack of detection methods to verify the origin of products makes the challenge even more complex.
New Zealand has a unique position as a major food exporter, and GE-free food is valued by the overseas markets we grow food for. It is vital that we continue to align with our trading partners and retain the ability to deliver premium GE-free food to respond to the ever-growing demand.
Therefore it is critical that active steps are taken now to:
- secure organic and GE-free seed stocks for future use in organics and GE-free production.
- prepare an action plan of how to keep the new GE organisms (and products derived from these) out of the organic chain of custody.
Dr Heli Matilainen, PhD in Biotechnology, MSc in Molecular Biology, has 10 years of hands-on experience in designing and generating GMOs. At present she is an organic advocate and founding director of Helix Organics (, www.helixorganics.co.nz), and a member of the IFOAM Technical Working Group for New Breeding Techniques.
IFOAM position on breeding techniques
An expert working group of IFOAM (International Federation of Organic Agriculture Movements) has drafted a position paper about which breeding techniques, including targeted genome engineering, are either compatible with organic systems or should be excluded, and what should be considered genetic engineering. This position paper – IFOAM Consultation on Breeding Techniques – was released for public consultation with feedback due by 31 March 2017.
Brookhouser, N, Raman, S, et al. 2017. May I cut in? Gene editing approaches in human induced pluripotent stem cells. Cells 6(1), 5.
Cong, L, Ran, FA, Cox, D, Lin, S. et al. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823.
Eckerstorfer M, Miklau, M, Gaugitsch, H. 2014. New Plant Breeding Techniques and Risks Associated with their Application. Environment Agency Austria, Report REP-0477, Umweltbundesamt GmbH, Vienna. ISBN 978-3-99004-282-3.
Fichtner, F. et al. 2014. Precision genetic modifications: New era in molecular biology and crop improvement. Planta, 239:921-39.
Fladung, M. 2016. Cibus’ herbicide-resistant canola in European limbo. Nat. Biotechnol. 34, 473–474.
Hilscher, J, et al. 2017. Targeted modification of plant genomes for precision crop breeding. Biotechnol. J. 12, 1600173.
Jeong YH, Kim Y, et al. 2016. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine beta-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination. Zygote, 24(3):442–456.
Kang, X, He, W, et al. 2016. Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas9- mediated genome editing. Journal of Assisted Reproduction and Genetics. 33(5):581-588.
Kim, H and Kim, J-S. 2014. A guide to genome engineering with programmable nucleases. Nature Reviews Genetics, Vol 15, pp.321-334.
Ledford, H. 2016. Riding the CRISPR wave. Nature, vol 531, pp. 156-159.
Lotti, S, et al. 2017. Modification of the genome of domestic animals, Animal Biotechnology, 19 Jan 2017.
Nishitani, C. et al. 2016. Efficient genome editing in apple using a CRISPR/Cas9 system. Sci. Rep. 6, 31481.
Puchta, H, and Fauser, F. 2013. Gene targeting in plants: 25 years later. Int. J. Dev. Biol. 57: 629-637.
Shan, Q., et al. 2015. Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. Plant Biotechnol. J. 13, 791–800.
Wang, Y, Cheng, X, et al. 2014. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947–951.
Walz, E. 2016. Gene-edited CRISPR mushroom escapes US regulation. Nature 532(7599), 293, 21 April 2016.
Photo credit: istock