TWN Info Service on Biosafety
23 November 2021
Third World Network
www.twn.my
Dear Friends and Colleagues
Genome Editing-Induced DNA Repair May Result in Unintended Outcomes
Genome editing encompasses new forms of genetic engineering techniques being increasingly applied to the development of agricultural and biomedical technologies, by ‘editing’ the DNA of living organisms, including plants, animals and human cells.
A new report examines the question of unintended ‘on-target’ effects that include various forms of genetic damage that scar edited genomes. This genetic damage is a common side-effect of the process of genome editing, resulting from error-prone mechanisms of DNA repair following genome editing induced DNA breaks, and the deployment of additional, less understood DNA repair pathways by the cell. Such unintended DNA repair outcomes will determine the efficiency and safety of gene-editing technologies applied to organisms.
The report concludes that genome edited organisms must be strictly regulated to allow for thorough characterisation of the full spectrum of unintended effects associated with the technology in the form of risk assessment and management, ensuring safety and efficacy of edited organisms. Furthermore, regulations are vital to ensure labelling and traceability of products in order to operationalise citizen’s and farmers’ rights to decide what to grow and consume, and to facilitate any recall/removal from the food chain and the environment following any unanticipated risk events.
With best wishes,
Third World Network
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ON-TARGET EFFECTS OF GENOME EDITING TECHNIQUES: (UN)REPAIRED DNA DAMAGE, A HINDERANCE TO SAFETY AND DEVELOPMENT?
GeneWatch
7 Oct 2021
http://www.genewatch.org/uploads/f03c6d66a9b354535738483c1c3d49e4/genome-editing-techniques-fin.pdf
[EXCERPTS ONLY]
Introduction
Genome editing encompasses new forms of genetic engineering techniques being increasingly applied to the development of agricultural and biomedical technologies, by ‘editing’ the DNA of living organisms (i.e. their ‘genome’), including plants, animals and human cells. The genome editing process involves the use of biological molecules (enzymes) which cut the DNA and various mechanisms which then repair it. Arguments over the safety and efficacy of genome editing techniques at the molecular level often hinge on aspects of their ‘precision’ in targeting DNA sequences of interest, and the ‘indistinguishability’ of engineered traits from mutations that may arise naturally.
Claims of genome editing precision or specificity have been repeatedly challenged by the accumulation of studies showing unintended effects, such as ‘off-target’ modification of additional regions of the genome to the ‘target site’ chosen by the developers. Evidence to date also reveals the issue of unintended ‘on-target’ effects that include various forms of genetic damage that scar edited genomes. This genetic damage is a common side-effect of the process of genome editing, resulting from error-prone mechanisms of DNA repair following genome editing induced DNA breaks, and the deployment of additional, less understood DNA repair pathways by the cell. However, unintended effects at the target site have received much less attention, and efforts to characterise and detect them are not routinely performed. Not only do such effects fundamentally challenge the notion of indistinguishability and safety, but they raise important questions regarding whether such side-effects are contributing to a bottleneck of gene edited crops reaching commercialisation.
What do genome editing tools actually do to DNA?
Common genome-editing tools, such as CRISPR-based systems, use chemicals (enzymes) called endonucleases which cut DNA. In most cases, they cut both strands of DNA, resulting in ‘double-strand breaks’. These genome editing tools are described as targeted and precise, because they can be engineered to cut a specific DNA sequence of choice. For example, they can be directed to cut a gene involved in the susceptibility of a plant to a herbicide, and thus aim to modify the gene to exert tolerance to a given herbicide. In the case of CRISPR systems, the targeted nature is conferred by guide RNA (gRNA) sequences complimentary to the target DNA sequence to be modified, which then direct the CRISPR enzyme (usually CRISPR/Cas9 endonuclease) (sometimes known as ‘DNA scissors’) to the target site. These guide RNAs are synthesised in the laboratory to target a sequence of interest and introduced to cells along with the rest of CRISPR machinery. This is usually delivered in the form of transgenic DNA constructs (i.e. constructs that include ‘transgenic’ or foreign DNA, from different organisms) that encode for CRISPR enzyme and the guide RNA. Other genome editing techniques such as TALENs, meganucleases (MNs) and zinc-finger nucleases (ZFNs), do not deploy gRNAs to target specific DNA sequences, but are instead protein-based enzymes that have DNA recognition sites to bind and cleave particular DNA sequences of interest. The initiation of DNA damage in the form of double-stranded breaks is the first step of the genome editing process. However, after this stage, the outcomes that result are not determined by the engineer but instead by the cell, which activates its own DNA repair machinery to repair the broken DNA. How the cell decides to repair the DNA will result in divergent outcomes, either unintended or intended.
As detailed below, while developers routinely make claims that this process is predictable, understood and well-defined, the complex outcomes of DNA repair processes are not completely understood, nor are they entirely controllable. Such uncertainties add further justifications to widespread calls to regulate genome edited technologies under legislation covering genetically modified organisms (GMOs), warranting careful scrutiny of the biosafety implications of genetic damage being documented in edited organisms and cells.
As detailed below, while developers routinely make claims that this process is predictable, understood and well-defined, the complex outcomes of DNA repair processes are not completely understood, nor are they entirely controllable. Such uncertainties add further justifications to widespread calls to regulate genome edited technologies under legislation covering genetically modified organisms (GMOs), warranting careful scrutiny of the biosafety implications of genetic damage being documented in edited organisms and cells (Agapito-Tenfen et al., 2018; Eckerstorfer et al., 2019; ENSSER, 2021; Testbiotech, 2021).
[….]
Genome edited crops must be regulated and assessed for unintended effects
In the field of medical research, unintended effects of genome editing are largely undisputed, as highlighted by recent reports on the state of play by prestigious medical organisations and researchers who warn against potential unintended effects that may result in diseases such as cancers. However, in other fields of genome editing research such as agriculture, the rates and implications of unintended effects are constantly challenged and dismissed by proponents, based on reductive understanding of DNA repair processes, and thus notions of precision and predictability of genome editing tools.
An illustrative example of on-target effects that support the case for strict characterisation and regulation of genome editing technologies, is the detection of foreign DNA unintentionally inserted into the genome of the recently developed genome edited ‘hornless’ cow. Instead of ‘editing’ the cow, an unintentional transgenic organism was generated by the capturing of unintentional DNA sequences to patch up the break site. The cow harbours DNA originating from the vector DNA, the vehicle used to deliver the DNA encoding for the editing machinery into the cells (including antibiotic resistance genes). The developers missed these unintended outcomes based on the incorrect assertion that integration events were not possible. Such a finding highlights the critical need for comprehensive molecular characterisation and analytical methods that allow for the detection of large-scale alterations that would otherwise be missed. Even in cases where some checks are done, the methods used are usually not sufficient to detect most unintended changes.
Unintended effects resulting in erroneous repair of DNA breaks are not routinely studied, and standard analytical protocols will miss large-scale on-target alterations. As recently observed in edited human cells, approximately 16 % of sampled cells had large unintended changes that would usually be missed with conventional detection protocols. It is thus worth referring to Agapito-Tenfen et al., (2018), with regard to the concept of indistinguishability being defined by someone’s choice of what to measure. What is chosen for knowing, also means choosing what remains unknown. The authors thus rightly highlight that new analytical methods are indeed undermining claims of indistinguishability. These methods should be deployed routinely for assessing edited organisms that may be released into the environment or food systems. Filling in these knowledge gaps also goes beyond biosafety assessment, but may also assist in enabling the traceability of commercialised food products that is required to ensure consumer choice and uphold regulatory decisions on unapproved gene edited organisms. Proper characterisation of on-target alterations can thus serve to assist in regulatory mechanisms to preserve traceability and consumer choice at the individual and national level.
Moreover, further understanding of these complex DNA repair outcomes is needed, as they also pertain to efficacy of this technology. While proponents regularly claim that genome editing is needed to generate useful traits for addressing serious societal problems of food insecurity and climate change, to date only two genome edited crops have been commercialised, suggesting potential bottlenecks in development of successful lines. The efficacy implications of unintended effects, including off-target changes and erroneous DNA repair outcomes, are yet to be fully understood. Further research is thus warranted before unsubstantiated claims about the benefits of genome editing technologies are used to rush through changes to GMO legislation that could remove requirements for important health and safety assessments.
Associating indistinguishability with safety also fails to acknowledge that such genome editing technologies, along with others (e.g. external RNA-based products, gene drive technologies and others that are moving engineering tools directly into the field), are increasing the magnitude and scale of human intervention. As highlighted by Heinemann et al. (2021), mutations introduced by genome editing or other genetic technologies, are not reliant on the processes of evolution, but instead can be driven by human activity, to ensure such mutations establish and spread in the environment. Genome editing is flexible and cheap, promoting widespread use, increasing the extent of genetic changes being pursued, and thus the likelihood of large-scale environmental introduction, with unknown consequences. Technologies that perform genetic engineering in the field, such as pollen-mediated, or viral-mediated delivery of genome editing machinery in the open environment, further confirm the need for thorough risk assessments and regulations.
Conclusions
Many questions remain regarding the mechanistic underpinnings of DNA repair pathways in the genome editing process, and how this may impact safety and efficacy of genome edited organisms:
- New and alternative DSB repair pathways have been shown to play a crucial role in DNA repair outcomes in a variety of species, with current understanding still evolving.
- Evidence to date clearly indicates that genome edited DNA repair outcomes are highly complex and variable, with a multitude of unintended effects that demonstrate a lack of ‘precision’, ‘efficiency’ and ‘indistinguishability’ from naturally arising mutations.
- Such unintended DNA repair outcomes will determine the efficiency and safety of gene-editing technologies applied to organisms.
- Complexity of unintended effects resulting from repair of genome-editing induced DNA damage remains an active field of research that is under acknowledged by those advocating for deregulation of genome editing technologies for food and environmental applications, yet remain uncontested in the medical field.
- The current categorization of gene-editing techniques into SDN1, SDN2 and SDN3 does not reflect the variety of pathways leading to genome editing outcomes and thus cannot be used to determine regulations of genome edited organisms, such as suggestions to exclude SDN-1 and SDN-2 applications from GMO legislation.
- Underestimation of on-target DNA changes leads to the under evaluation and analysis of unintended effects that need to be systematically characterised to ensure safety prior to the release of genome edited organisms into the environment.
- Genome edited organisms must be strictly regulated to allow for thorough characterisation of the full spectrum of unintended effects associated with the technology in the form of risk assessment and management, ensuring safety and efficacy of edited organisms. Further, regulations are vital to ensure labelling and traceability of products in order to operationalise citizen and farmer rights to decide what to grow and consume, and to facilitate any recall/removal from the food chain and the environment following any unanticipated risk events.