Detection Methods Fit-for-Purpose in Enforcement Control of Genetically Modified Plants Produced with Novel Genomic Techniques (NGTs)
Round 1
Reviewer 1 Report
I accepted to review this perspective paper on the use of NGT for detecting GMOs, although I have not a strong expertise in this field. The paper is clearly written, but I feel that some parts are quite redundant, with the discussion and conclusion reminding some details explained in previous parts. I think this paper could be shortened.
The distinction between GMOs produced by genome editing and spontaneous mutants could be a strong challenge that is not clearly presented. I consider that a single nucleotide polymorphism (such as a SNP) that modifies an agronomic trait, could also appear spontaneously and be selected by breeders; in this case, it's hard (and maybe impossible) to make the distinction between GMOs and spontaneous mutants. So, I suggest that the authors could be more precise on the nature of the genetic modification. A single nucleotide variant (SNV) could also be an indel that will disrupt gene function, and such a SNV is easier to identify since the probability that the same mutation spontaneously occurred is very unlikely (to not say impossible).
Then, I feel that the topic of this perspective answers to a "technocratric demand". I agree that legal aspects need to be addressed because there are some important ecological and economic challenges with the use of GMOs. However, I think that many problems of GMOs are first linked to the limited education of producers and consumers, who are still not able to evaluate putative problems related to the use and consumption of such GMOs. As an example, the "EU regulatory framework" aims first at preventing damage to human health before considering the consequences on economic sectors and mostly on the environment, especially the biodiversity erosion that is rarely (and even never) mentioned. Awareness is really needed, and these should be the first issues to be pointed out. A couple of sentences in the introduction/abstract considering these aspects might be welcome...
Author Response
R: Reviewer’s comments
A: Authors’ response
R: I accepted to review this perspective paper on the use of NGT for detecting GMOs, although I have not a strong expertise in this field. The paper is clearly written, but I feel that some parts are quite redundant, with the discussion and conclusion reminding some details explained in previous parts. I think this paper could be shortened.
A: Thank you very much for your valuable suggestion to improve the manuscript. Taking this into consideration, we have eliminated redundancies and have tried to streamline the main messages.
R: The distinction between GMOs produced by genome editing and spontaneous mutants could be a strong challenge that is not clearly presented. I consider that a single nucleotide polymorphism (such as a SNP) that modifies an agronomic trait, could also appear spontaneously and be selected by breeders; in this case, it's hard (and maybe impossible) to make the distinction between GMOs and spontaneous mutants. So, I suggest that the authors could be more precise on the nature of the genetic modification . A single nucleotide variant (SNV) could also be an indel that will disrupt gene function, and such a SNV is easier to identify since the probability that the same mutation spontaneously occurred is very unlikely (to not say impossible).
A: We agree that it is indeed challenging to distinguish between genome-edited and spontaneously occurring mutations. The manuscript describes the technical background of methods that can be currently implemented in enforcement laboratories, and limitations and challenges that enforcement laboratories face concerning relevant technical requirements. The manuscript, however, does not discuss the challenges related to distinguishing the (technical) origin of DNA alterations. We describe that detection in the narrow sense is technically feasible. Identification is an issue that may be solved by detection methodology given that adequate information is available and the sequence alteration(s) is unique. This is the case for GMOs, but for genome-edited plants this can only be assessed on a case-by-case basis. We agree that for certain mutations detection might also allow to identify a genome-edited plant. The underlying genome editing technique, however, is even more challenging to identify.
R: Then, I feel that the topic of this perspective answers to a "technocratic demand ". I agree that legal aspects need to be addressed because there are some important ecological and economic challenges with the use of GMOs. However, I think that many problems of GMOs are first linked to the limited education of producers and consumers, who are still not able to evaluate putative problems related to the use and consumption of such GMOs. As an example, the "EU regulatory framework" aims first at preventing damage to human health before considering the consequences on economic sectors and mostly on the environment, especially the biodiversity erosion that is rarely (and even never) mentioned. Awareness is really needed, and these should be the first issues to be pointed out. A couple of sentences in the introduction/abstract considering these aspects might be welcome...
A: Enforcement encompasses official control activities (in particular implementation of labelling requirements, controlling the presence of unauthorised GMOs on the market) based on the European GMO regulatory framework but is not involved in its definition. Although we agree that it might be worthwhile to consider some additional aspects of the GMO legislation, the focus of the manuscript at hand is not to point to possible ecological and economic challenges or other elements potentially worth discussing. We feel that adding such messages to the text but not discussing them in depth (due to the manuscript’s focus) would not improve the manuscript.
Reviewer 2 Report
The review is an interesting read and provides information about existing GMO detection methodologies and procedures.
I am not convinced that the central theme is addressed in terms of describing fit-for-purpose detection methods for plants harboring small NGT-derived events. It is clear that unknown changes cannot be detected and so the condition on ln58 "is pivotal to comply with the requirements of the EU regulatory framework for GMO's" cannot be met.
Whilst it is clear, as described in recent publications, that suitable assays can developed for a known event this is unlikely to be the main challenge with NGT-derived material in the future.
If, as anticipated, there is widespread global uptake of NGT-derived crops there are significant challenges. Although it could be argued that multiplexing of a large number of assays could detect a range of known edits used in a particular crop there is no methodology to detect unknown ones.
With many countries not regulating some forms of NGT-derived crops and these unregulated crops over time being used in breeding programmes detection will become increasingly challenging.
The case where an existing natural mutation in one variety is introduced into another using NGT technology the two varieties auld be undistinguishable even with a suitable assay.
Even if it were possible to detect undisclosed changes it would not be possible to determine how these arose.
This is hinted at in the paragraph beginning on ln247 with mention of the need for appropriate reference material but not made explicit.
I believe it is essential to make explicit the limitations associated with detection. Otherwise a reader could get the somewhat misleading impression that there is an effective method to detected undeclared NGT-derived crops.
The manuscript could also be shortened with no loss of impact. There is great deal of repetition with the same material repeated in different sections e.g. almost the same statement in the discussion ln197 as earlier ln87.
Author Response
R: Reviewer’s comments
A: Authors’ response
R: The review is an interesting read and provides information about existing GMO detection methodologies and procedures.
I am not convinced that the central theme is addressed in terms of describing fit-for-purpose detection methods for plants harboring small NGT-derived events. It is clear that unknown changes cannot be detected and so the condition on ln58 "is pivotal to comply with the requirements of the EU regulatory framework for GMO's" cannot be met.
A: Thank you very much for pointing out the discrepancy between the enforcement reality and compliance with the requirements of the EU GMO regulatory framework. We agree that not all issues are resolved concerning the preconditions required to be able to implement all relevant regulations. Accordingly, we have modified the sentence.
The focus of the manuscript is the analysis of methods “fit-for-purpose” in the sense that 1) the methods are suitable to detect SNVs and 2) considering the current technical resources and expertise of staff. In this context, we make a clear difference between “detection” and “identification”. We describe that detection in the narrow sense is possible. Additionally, we discuss the specific requirements from an enforcement laboratory’s point of view. To our knowledge, these issues have to date not been presented in a paper. Notwithstanding technical possibilities of detection, in the context of enforcement additional standards need to be complied with, e.g. validation and accreditation.
Concerning the unknown changes, whenever screening elements are not present detection is seriously hampered, regardless of the underlying technique leading to DNA alterations. This is the case for GMOs for which no information is available and thereby does not differ in the case of genome-edited plants. This aspect is considered in the manuscript.
R: Whilst it is clear, as described in recent publications, that suitable assays can developed for a known event this is unlikely to be the main challenge with NGT-derived material in the future.
If, as anticipated, there is widespread global uptake of NGT-derived crops there are significant challenges. Although it could be argued that multiplexing of a large number of assays could detect a range of known edits used in a particular crop there is no methodology to detect unknown ones.
With many countries not regulating some forms of NGT-derived crops and these unregulated crops over time being used in breeding programmes detection will become increasingly challenging.
A: As stated, the major focus of the manuscript is the assessment of methods fit-for-purpose in a specific sense. We agree that detection methods per se do not solve all the issues related to the challenges in the context of NGT material on the market. We hope that the modifications of the text clarify that these limitations exist.
R: The case where an existing natural mutation in one variety is introduced into another using NGT technology the two varieties would be undistinguishable even with a suitable assay.
Even if it were possible to detect undisclosed changes it would not be possible to determine how these arose.
A: We agree that it is indeed challenging to distinguish between genome-edited and spontaneously occurring mutations. The manuscript describes the technical background of methods that can be currently implemented in enforcement laboratories, and limitations and challenges that enforcement laboratories face concerning relevant technical requirements. The manuscript, however, does not discuss the challenges related to distinguishing the (technical) origin of DNA alterations. We describe that detection in the narrow sense is technically feasible. Identification is an issue that may be solved by detection methodology given that adequate information is available and the sequence alteration(s) is unique. This is the case for GMOs but for genome-edited plants this can only be assessed on a case-by-case basis. We agree that for certain mutations detection might also allow to identify a genome-edited plant. The underlying genome editing technique, however, is even more challenging to identify. We have modified the text accordingly and hope that this sufficiently explains the situation.
R: This is hinted at in the paragraph beginning on ln247 with mention of the need for appropriate reference material but not made explicit.
A: We explain that in the context of enforcement control reference material is needed for diverse purposes. We hope that, as explained above, the issue of distinguishing different causes of mutations is discussed adequately in the modified manuscript.
R: I believe it is essential to make explicit the limitations associated with detection. Otherwise a reader could get the somewhat misleading impression that there is an effective method to detected undeclared NGT-derived crops.
A: The abstract has been modified accordingly to avoid a misleading impression. Also, the focus of the manuscript and its conclusions have been specified.
R: The manuscript could also be shortened with no loss of impact. There is great deal of repetition with the same material repeated in different sections e.g. almost the same statement in the discussion ln197 as earlier ln87.
A: Thank you very much for your valuable suggestion to improve the manuscript. Taking this into consideration, we have eliminated redundancies and have tried to streamline the main messages.
Round 2
Reviewer 2 Report
I find the modifications to the manuscript have improved its readability and it is now clearer that the methods used will only detect known genome-edited changes.
I think it would be beneficial to include the word known in the sentence beginning on line 161 that reference the Chhalliyil et al reference.
i.e. a detection method for a known single base pair edit (20).
This will alleviate concerns in the scientific community around the referenced publications wide claims about the applicability of the described assay.
The manuscript could include a sentence to reflect the the the prerequisite information described as required on line 395-397, and supplied by developers, may not be available form developers going forward. It seems likely if genome editing is widely taken up in countries where it is not regulated as a GMO such unregulated products will enter international supply chains.
the sentence ln 395 needs editing .... to develop to detect needs editing.
in the references on ln 497 edited is spelt incorrectly.
The references are in a number of different formats or incomplete?
in some year is bolded some not etc.
Author Response
R: Reviewer’s comments
A: Authors’ response
R: I find the modifications to the manuscript have improved its readability and it is now clearer that the methods used will only detect known genome-edited changes.
R: I think it would be beneficial to include the word known in the sentence beginning on line 161 that reference the Chhalliyil et al reference.
i.e. a detection method for a known single base pair edit (20).
This will alleviate concerns in the scientific community around the referenced publications wide claims about the applicability of the described assay.
A: We agree with the Reviewer and have included the suggested word; please refer to line 162 of the revised manuscript.
R: The manuscript could include a sentence to reflect the the the prerequisite information described as required on line 395-397, and supplied by developers, may not be available form developers going forward. It seems likely if genome editing is widely taken up in countries where it is not regulated as a GMO such unregulated products will enter international supply chains.
A: Please refer to lines 400-404, where this aspect is reflected in the revised text.
R: the sentence ln 395 needs editing .... to develop to detect needs editing.
A: Thank you! In the course of revision, the word “methods” had been deleted by mistake. We have rephrased the sentence by including it again (line 397, revised manuscript).
R: in the references on ln 497 edited is spelt incorrectly.
A: Thank you! The spelling mistake has been corrected (line 506).
R: The references are in a number of different formats or incomplete?
in some year is bolded some not etc.
A: We have paid attention to the MDPI style when formatting the manuscript. The references have been checked for completeness.
