List compiled by GMWatch. Technical advisor: Dr Michael Antoniou
More papers have been published on unintended outcomes and risks of gene editing in medical research on human and animal cells and laboratory animals, compared with plants. These results have implications for the gene editing of farm animals. Moreover, the problems found with human and animal gene editing are increasingly being confirmed in plant gene editing. The studies listed below are on human and animal cells, laboratory animals, livestock animals, and plants.
The unintended mutational (DNA damaging) outcomes summarized below occur after the gene-editing tool has completed its task of creating a double-strand DNA break. The mutations occur as a consequence of the cell’s DNA repair machinery, over which the genetic engineer has no control. So even if scientists eventually succeed in avoiding off-target mutations, most of the unintended mutations described can still occur at the intended gene-editing site.
This lack of full control of the gene-editing procedure, as well as gaps in our knowledge of outcomes, point to the need for strict regulation of gene editing in food crops and farm animals. Regulation must start from consideration of the genetic engineering process used to create the gene-edited organism (“process-based regulation”), so that regulators know where things can go wrong and what to look for.
NEED FOR REGULATION
New GM plants do not have a history of safe use and should not be exempted from biosafety assessments.
Koller F and Cieslak M (2023). A perspective from the EU: unintended genetic changes in plants caused by NGT — their relevance for a comprehensive molecular characterisation and risk assessment. Front. Bioeng. Biotechnol. 11. 27 October. Sec. Biosafety and Biosecurity. https://doi.org/10.3389/fbioe.2023.1276226
Chu P and Agapito-Tenfen SZ (2022). Unintended genomic outcomes in current and next generation GM techniques: A systematic review. Plants 2022, 11, 2997. https://pubmed.ncbi.nlm.nih.gov/36365450/
Eckerstorfer MF et al (2021). BioTech 10(3). https://doi.org/10.3390/biotech10030010
Kawall K (2021). Plants 10(11). 10.3390/plants10112259
https://www.mdpi.com/2223-7747/10/11/2259/htm
Eckerstorfer MF et al (2019). Front Bioeng Biotechnol 7:31. https://www.frontiersin.org/articles/10.3389/fbioe.2019.00031/full
Gelinksky E and Hilbeck A (2018). Environ Sci Europe 30(1):52.
https://enveurope.springeropen.com/articles/10.1186/s12302-018-0182-9
Kawall K et al (2020). Environmental Sciences Europe 32, Article no 106 (2020) https://enveurope.springeropen.com/articles/10.1186/s12302-020-00361-2
CHANGES INDUCED BY GENE EDITING ARE NOT THE SAME AS HAPPENS IN NATURE
Gene editing makes the whole genome accessible for changes – unlike naturally occurring genetic changes.
Koller F and Cieslak M (2023). A perspective from the EU: unintended genetic changes in plants caused by NGT — their relevance for a comprehensive molecular characterisation and risk assessment. Front. Bioeng. Biotechnol. 11. 27 October. Sec. Biosafety and Biosecurity. https://doi.org/10.3389/fbioe.2023.1276226
Kawall K (2019). Frontiers in Plant Science 10:525. https://www.frontiersin.org/articles/10.3389/fpls.2019.00525/full
UNINTENDED MUTATIONS
Below is a selection of studies showing different types of unintended mutations resulting from gene editing that can affect the functioning of multiple gene systems. The consequences are an alteration in the plant’s protein and biochemical function, which could lead to poor crop performance and/or the production of novel toxins and allergens or higher levels of existing toxins and allergens.
Off-target mutations
Gene-editing tools, especially CRISPR, are prone to causing mutations (damage) to the organism’s DNA at locations other than the intended edit site ("off-target mutations"). This can alter the function of other genes, with unknown consequences to biochemical composition and function.
Wolt JD et al (2016). The Plant Genome 9(3): 10.3835/plantgenome2016.05.0047. https://acsess.onlinelibrary.wiley.com/doi/full/10.3835/plantgenome2016.05.0047
Zhu C et al (2017). Trends in Plant Science 22(1):38–52. https://www.ncbi.nlm.nih.gov/pubmed/27645899
Large deletions and rearrangements of DNA at both off-target and on-target gene editing sites
Large deletions and rearrangements of the plant’s genome, which can involve thousands of base units of DNA, have been observed following CRISPR gene editing. These mutations can affect the functioning of many genes, leading to alterations in the plant’s protein and biochemical composition.
Biswas S et al (2020). Journal of Genetics and Genomics. May 21. https://www.sciencedirect.com/science/article/pii/S1673852720300916
Höijer I et al (2021). bioRxiv. doi: https://doi.org/10.1101/2021.10.05.463186. https://www.biorxiv.org/content/10.1101/2021.10.05.463186v1
Kosicki M et al (2018). Nature Biotechnology 36:765–771. https://www.nature.com/articles/nbt.4192
Mou H et al. (2017). Genome Biology 18:108. https://genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1237-8
Shin HY et al. (2017). Nature Communications 8, 15464. https://www.ncbi.nlm.nih.gov/pubmed/28561021
Genomic rearrangement resulting from shattering of chromosomes (chromothripsis) at on-target gene editing sites
CRISPR gene editing for gene therapy applications can lead to massive damage to chromosomes. The phenomenon is known as chromothripsis. The fact that the damage occurs "on-target" – at the intended edit site – means that any attempts to target the CRISPR gene editing more precisely will not solve this problem. The study by Samach A et al (2023) below revealed chromothripsis-like effects after the application of CRISPR/Cas gene editing in the genome of tomatoes.
Leibowitz ML et al (2021). Nat Genet. 53(6):895-905. doi: 10.1038/s41588-021-00838-7. Epub 2021 Apr 12. https://pubmed.ncbi.nlm.nih.gov/33846636/
Samach A et al (2023). bioRxiv, 24 May. https://www.biorxiv.org/content/10.1101/2023.05.22.541757v1.
Creation of new gene sequences leads to new RNA and protein products
Alteration of the genetic code of the targeted gene can produce mutant forms of the protein it encodes for, new RNA, and new protein products. These outcomes can lead to changes in the plant’s biochemistry.
Mou H et al. (2017). Genome Biology 18:108. https://genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1237-8
Tuladhar R et al (2019). Nat Commun 10, 4056 (2019). https://www.nature.com/articles/s41467-019-12028-5
Smits AH et al (2019). Nat Methods 16, 1087–1093. https://www.nature.com/articles/s41592-019-0614-5
Gene-editing process-induced mutations
The gene editing process, taken as a whole (including plant tissue culture and GM transformation procedure), induces hundreds of unintended mutations throughout the genome of the plant. This can affect multiple gene functions with unknown consequences to protein biochemistry and metabolic activity.
Tang X et al (2018). Genome Biology 19:84. https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1458-5
Insertion of foreign and contaminating DNA into genome at editing sites
Following creation of a double-strand DNA break by the CRISPR gene-editing tool, the repair can unexpectedly include the insertion and rejoining of the broken DNA ends of the recombination template DNA used in SDN-2 and -3, or the insertion of contaminating DNA present in materials used in the plant tissue culture. This insertion of extraneous DNA in the genome of the plant, which can take place at off-target sites as well as the intended on-target editing site, has the effect of introducing new gene functions, as well as disrupting the function of host genes. These effects can combine to alter the biochemical function of the plant in unexpected ways.
Reports (Norris et al., 2020; Skryabin et al., 2020; Molteni 2020) describe insertion of the whole plasmid DNA molecules that acted as the recombination template for the SDN-2 or SDN-3 procedure. The insertion of these plasmid DNA templates will invariably result in at least one antibiotic resistance gene being incorporated in the genome, as these are a component of plasmids. This risks the transfer of antibiotic resistance genes to disease-causing bacteria in the environment and more worryingly, in the gut of the consumer, which would compromise medical use of antibiotics.
Norris AL et al (2020). Nat Biotech 38(2):163-164. https://www.nature.com/articles/s41587-019-0394-6
MEDIA ARTICLE: Molteni M (2020). WIRED, 24 July. https://www.wired.com/story/a-crispr-calf-is-born-its-definitely-a-boy/
Skryabin BV et al. (2020). Science Advances 6(7), eaax2941. https://advances.sciencemag.org/content/6/7/eaax2941
Ono R et al (2019). Communications Biology 2: 57. https://www.nature.com/articles/s42003-019-0300-2.pdf?origin=ppub
For more details on individual studies, see: https://www.gmwatch.org/en/news/latest-news/19223