Literature horizon scan for new scientific data on plants, microorganisms and animals, and their products obtained by new genomic techniques (October 2025)

TL;DR

This report summarizes a literature scan on new scientific data related to plants, microorganisms, and animals modified using new genomic techniques, and finds no new risks.

Contribution

A systematic literature scan and evaluation of new scientific data on organisms modified by new genomic techniques.

Findings

No new hazards or risks were identified in the retrieved studies.

Existing EFSA guidelines remain applicable for assessing risks from new genomic techniques.

The report outlines limitations of the search and suggests improvements for future scans.

Abstract

The European Food Safety Authority has issued several scientific opinions on plants, microorganisms and animals obtained through certain new genomic techniques (NGTs), following requests received by the European Commission. These scientific opinions provided considerations on the potential risks associated with NGTs, as compared to conventional breeding techniques and established genomic techniques (EGTs), and on the applicability of existing guidelines for the risk assessment of plants, microorganisms and animals obtained by NGTs and products thereof. Against this background, EFSA was asked by the European Commission ‘to provide scientific and technical assistance for a regular horizon scanning to assess new scientific data on plants, microorganisms and animals, and products thereof obtained by new genomic techniques’, to assess any new data and evidence emerging from these studies, and to consider whether it may have implications for EFSA's relevant scientific opinions. A critical assessment of the quality and relevance of these studies should also be conducted, and biannual reports delivered to the Commission. This report presents the outcome of the literature search on new scientific data on plants, microorganisms and animals and products thereof obtained by NGTs, performed as described in the protocol made available for public consultation in May–June 2025. The report describes search strategies and inclusion/exclusion criteria applied for literature review and presents the results of the assessment performed for new scientific data (published up to October 2025) on plants, microorganisms and animals and products thereof obtained by NGTs. The report discusses the limitations of the search and provides recommendations for improvement. EFSA concluded that none of the studies retrieved by the literature search contained new hazards or risks not previously considered in EFSA scientific opinions.

Tables

TABLE 1. EFSA scientific opinions relevant for each AQ.

References	Title
AQ1 (NGTs applied to plants)
EFSA GMO Panel (2012a)	EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2012a). Scientific Opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site‐Directed Nucleases with similar function. EFSA Journal, 10(10), 2943. https://doi.org/10.2903/j.efsa.2012.2943
EFSA GMO Panel (2012b)	EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2012b). Scientific Opinion addressing the safety assessment of plants developed through cisgenesis and intragenesis. EFSA Journal, 10(2), 2561. https://doi.org/10.2903/j.efsa.2012.2561
EFSA GMO Panel (2020)	EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2020). Applicability of the EFSA Opinion on SDNs type 3 for the safety assessment of plants developed using SDNs type 1 and 2 and oligonucleotide‐directed mutagenesis. EFSA Journal, 18(11), 6299. https://doi.org/10.2903/j.efsa.2020.6299
EFSA GMO Panel (2021)	EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2021). Scientific Opinion on the evaluation of existing guidelines for their adequacy for the molecular characterisation and environmental risk assessment of genetically modified plants obtained through synthetic biology. EFSA Journal, 19(2), 6301. https://doi.org/10.2903/j.efsa.2021.6301
EFSA GMO Panel (2022a)	EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2022a). Updated scientific opinion on plants developed through cisgenesis and intragenesis. EFSA Journal, 20(10), 7621. https://doi.org/10.2903/j.efsa.2022.7621
EFSA GMO Panel (2022b)	EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2022b). Scientific Opinion on the evaluation of existing guidelines for their adequacy for the food and feed risk assessment of genetically modified plants obtained through synthetic biology. EFSA Journal, 20(7), 7410. https://doi.org/10.2903/j.efsa.2022.7410
EFSA GMO Panel (2022c)	EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms). (2022c). Statement on criteria for risk assessment of plants produced by targeted mutagenesis, cisgenesis and intragenesis. EFSA Journal, 20(10), 7618. https://doi.org/10.2903/j.efsa.2022.7618
AQ2 (NGTs applied to microorganisms)
EFSA Scientific Committee (2020)	EFSA Scientific Committee, More, S., Bampidis, V., Benford, D., Bragard, C., Halldorsson, T., Hernández‐Jerez, A., Hougaard Bennekou, S., Koutsoumanis, K., Machera, K., Naegeli, H., Nielsen, SS., Schlatter, J., Schrenk, D., Silano, V., Turck, D., Younes, M., Glandorf, B., Herman, L., Tebbe, C., Vlak, J., Aguilera, J., Schoonjans, R. and Cocconcelli, P.S. (2020). Scientific Opinion on the evaluation of existing guidelines for their adequacy for the microbial characterisation and environmental risk assessment of microorganisms obtained through synthetic biology. EFSA Journal, 18(10), 6263. https://doi.org/10.2903/j.efsa.2020.6263
EFSA Scientific Committee (2022)	EFSA Scientific Committee, More, S., Bampidis, V., Benford, D., Bragard, C., Halldorsson, T., Hernández‐Jerez, A., Bennekou, S.H., Koutsoumanis, K., Lambré, C., Machera, K., Mullins, E., Nielsen, S.S., Schlatter, J., Schrenk, D., Turck, D., Younes, M., Herman, L., Pelaez, C., van Loveren, H., Vlak, J., Revez, J., Aguilera, J., Schoonjans, R. and Cocconcelli, P.S. (2022). Eevaluation of existing guidelines for their adequacy for the food and feed risk assessment of microorganisms obtained through synthetic biology. EFSA Journal, 20(8), 7479. https://doi.org/10.2903/j.efsa.2022.7479
EFSA GMO Panel (2024)	EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Mullins, E., Bresson, J.‐L., Dewhurst, I. C., Epstein, M. M., Firbank, L. G., Guerche, P., Hejatko, J., Moreno, F. J., Naegeli, H., Nogué, F., Rostoks, N., Sánchez Serrano, J. J., Savoini, G., Veromann, E., Veronesi, F., Cocconcelli, P. S., Glandorf, D., Herman, L., … Dalmay, T. (2024). New developments in biotechnology applied to microorganisms. EFSA Journal, 22(7), 8895. https://doi.org/10.2903/j.efsa.2024.8895
AQ3 (NGTs applied to animals)
EFSA GMO Panel (2025)	EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Casacuberta, J., Barro, F., Braeuning, A., de Maagd, R., Epstein, M. M., Frenzel, T., Gallois, J.‐L., Koning, F., Messéan, A., Moreno, F. J., Nogué, F., Schulman, A. H., Tebbe, C., Veromann, E., Firbank, L., Glandorf, D., Herskin, M. S., Lillico, S. G., … Savoini, G. (2025). New developments in biotechnology applied to animals: An assessment of the adequacy and sufficiency of current EFSA guidance for animal risk assessment. EFSA Journal, 23(8), 9566. https://doi.org/10.2903/j.efsa.2025.9566

TABLE 2. Time ranges and dates of literature searches performed using three different sources of evidence.

AQ	Time range for the search	Date of search
AQ1	01/01/2022–onwards	Scopus: 30/9/2025 Web of Science: 29/9/2025 Pubmed: 7/10/2025
AQ2	01/01/2024–onwards	Scopus: 3/10/2025 Web of Science: 3/10/2025 Pubmed: 3/10/2025
AQ3	01/01/2025–onwards	Scopus: 22/10/2025 Web of Science: 22/10/2025 Pubmed: 22/10/2025

TABLE 3. Number of publications retrieved after each step of the literature search.

Review step	No. of publications
	AQ1 (NGTs applied to plants)	AQ2 (NGTs applied to microorganisms)	AQ3 (NGTs applied to animals)
Retrieved by the literature search (after de‐duplication)	1021 a	2114	1377
Excluded after title/abstract screening	982	2069	1339
Included after title/abstract screening	39	45	38
Excluded after full‐text screening b	34	35	30
Language	2	0	0
Organisms	1	4	0
Outcome/conclusions	29	29	26
Publication type	4	2	2
Scope/intended use	2	19	3
Techniques	5	1	2
Other	0	2	0
Included after full‐text screening c	5	10	8

TABLE 4. Reference publications used to identify and validate search terms.

Reference	AQ	Title
Bessoltane et al. (2022)	AQ1	Bessoltane, N., Charlot, F., Guyon‐Debast, A., Charif, D., Mara, K., Collonnier, C., Perroud, P. F., Tepfer, M., and Nogué, F. (2022). Genome‐wide specificity of plant genome editing by both CRISPR–Cas9 and TALEN. Scientific Reports, 12, 9330. https://doi.org/10.1038/s41598‐022‐13034‐2
Liu et al. (2023)	AQ1	Liu, J., Wang, F. Z., Li, C., Li, Y., and Li, J. F. (2023). Hidden prevalence of deletion‐inversion bi‐alleles in CRISPR‐mediated deletions of tandemly arrayed genes in plants. Nature Communications, 14, 6787. https://doi.org/10.1038/s41467‐023‐42490‐1
Samach et al. (2023)	AQ1	Samach, A., Mafessoni, F., Gross, O., Melamed‐Bessudo, C., Filler‐Hayut, S., Dahan‐Meir, T., Amsellem, Z., Pawlowski, W. P., and Levy, A. A. (2023). CRISPR/Cas9‐induced DNA breaks trigger crossover, chromosomal loss, and chromothripsis‐like rearrangements. Plant Cell, 35(11), 3957–3972. https://doi.org/10.1093/plcell/koad209
Xiong et al. (2023)	AQ1	Xiong, X., Liu, K., Li, Z, Xia, F. N., Ruan, X. M., He. X., and Li, J. F. (2023). Split complementation of base editors to minimize off‐target edits. Nature Plants, 9, 1832–1847. https://doi.org/10.1038/s41477‐024‐01777‐x
Cui et al. (2025)	AQ1	Cui, S., Liu, H., Tian, L., Dong, X., Li, X., and Qu, L. Q. (2025). Generation and Nutritional Evaluation of Intragenic Rice with α‐Linolenic Acid Hyperfortified in Seeds. Journal of Agricultural Food Chemistry, https://doi.org/10.1021/acs.jafc.5c05281
EFSA GMO Panel (2024	AQ2	EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Mullins, E., Bresson, J.‐L., Dewhurst, I. C., Epstein, M. M., Firbank, L. G., Guerche, P., Hejatko, J., Moreno, F. J., Naegeli, H., Nogué, F., Rostoks, N., Sánchez Serrano, J. J., Savoini, G., Veromann, E., Veronesi, F., Cocconcelli, P. S., Glandorf, D., Herman, L., … Dalmay, T. (2024). New developments in biotechnology applied to microorganisms. EFSA Journal, 22(7), e8895. https://doi.org/10.2903/j.efsa.2024.8895
Mahdi et al. (2025)	AQ3	Mahdi, A. K., Fitzpatrick, D. S., Hagen, D. E., McNabb, B. R., Urbano Beach, T., Muir, W. M., Werry, N., Van Eenennaam, A. L., Medrano, J. F., and Ross, P. J. (2025). Efficient Generation of SOCS2 Knock‐Out Sheep by Electroporation of CRISPR‐Cas9 Ribonucleoprotein Complex with Dual‐sgRNAs. CRISPR Journal, 8(1):13–25. https://doi.org/10.1089/crispr.2024.0055

TABLE 5. Number of publications retrieved for AQ1, divided by publication date.

Review step	No. of publications
	Publication date: 2022	Publication date: 2023	Publication date: 2024	Publication date: 2025
Retrieved by the literature search (after de‐duplication)	133	118	181	589
Included after title/abstract screening	5	2	2	31
Included after full‐text screening	0	0	0	5

TABLE 6. Publications retrieved for AQ2 divided by use of the term “human” in the search.

Review step	No. of publications
	“Human” included in search terms	“Human” excluded in search terms	Used for calibration exercise	Moved from another search string a
Retrieved by the literature search (after de‐duplication)	626	1377	100	11
Included after title/abstract screening	2	41	2	0
Included after full‐text screeningc	0	10	0	0

TABLE 7. Publications retrieved for AQ3 divided by the species used in the search.

Review step	No. of publications
	Agri‐food animals	Model animals	Used for calibration exercise	Moved from another search string a
Retrieved by the literature search (after de‐duplication)	272	975	100	30
Included after title/abstract screening	24	10	4	0
Included after full‐text screening	5	3	0	0

TABLE 8. Publications re‐assigned to another group after the title/abstract screening.

		Search on group of organisms in which the publications were retrieved
		Plants (AQ1)	Microorganisms (AQ2)	Animals (AQ3)
Group of organisms for which the publications were potentially relevant	Plants (AQ1)	N/A	1	0
	Microorganisms (AQ2)	10	N/A	1
	Animals (AQ3)	5	25	N/A

Full text

INTRODUCTION

1

Background and Terms of Reference (ToR) as provided by the requestor

1

1.1

Background

1.1.1

Given the rapid development of new genomic techniques (NGTs) and their potential applications, new scientific studies are regularly published regarding the development and current or potential market presence of NGTs in plants, animals and microorganisms. These studies can contain new scientific data and evidence relevant to the safety, risk assessment and other considerations made by EFSA in its adopted or upcoming scientific opinions on NGTs applied to plants, microorganisms and animals.

Beyond the extensive literature reviews conducted by EFSA in response to various mandates in this domain, EFSA has also independently analysed new evidence related to its scientific work on organisms obtained by NGTs. In addition, the European Commission (EC) has requested EFSA to consider individual scientific publications brought to its attention by stakeholders on an ‘ad‐hoc’ basis. For reasons of completeness and transparency, a more formal and structured approach, including biannual reporting to the EC, is deemed necessary.

Terms of Reference as provided by the requestor

1.1.2

In light of the above, in accordance with Article 31 of Regulation (EC) No 178/2002, EFSA is requested to regularly screen the scientific literature to identify relevant studies for the assessment of food and feed safety and the environmental safety of organisms and products obtained with NGTs, to assess any new evidence emerging from these studies, and to consider whether it may have implications for EFSA's relevant scientific opinions. A critical assessment of the quality and relevance of these studies should also be conducted, and regular reports delivered to the Commission.

This request does not include evidence assessed in adopted and upcoming opinions.

More specifically, EFSA is requested to:

1. Develop and validate search strategies to be used for searching of scientific literature databases with adequate sensitivity and specificity to identify studies that could be relevant as regards the safety, risk assessment and other considerations examined by EFSA in its relevant scientific opinions concerning NGTs applied to plants, microorganisms and animals.
2. Develop and pilot criteria for evidence inclusion and exclusion, and critical appraisal tools to support the assessment of relevant published scientific evidence.
3. Extract and summarise relevant data and evidence and assess whether any new data and evidence may have implications for EFSA relevant scientific opinions.
4. Deliver biannual reports on relevant findings.

Interpretation of the Terms of Reference

1.2

EFSA interprets the overall objective of the mandate as follows:

To identify and assess new evidence relevant for the safety, risk assessment and other considerations examined by EFSA in its scientific opinions on NGTs applied to plants, microorganisms and animals.

This objective was translated into assessment questions (AQs) and sub‐questions (SQs) as detailed in the literature search protocol (Annex A). The protocol describes the search strategies, inclusion and exclusion criteria, methodology for evidence appraisal and lists the ‘EFSA's relevant scientific opinions’ defined by the mandate, together with their conclusions (Appendix A to the protocol). The draft protocol was shared for public consultation.

To address the mandate, the following NGT definition was used: techniques capable to change the genetic material of an organism and that have emerged or have been developed since 2001, when the existing GMO legislation 2 was adopted. This definition was used by the European Commission in ‘Study on the status of new genomic techniques under Union law and in light of the Court of Justice ruling in Case C‐528/163’.

This report is the second biannual report requested in ToR4, describes the pilot search methodology (ToR1 and ToR2) for AQ2 and AQ3, and presents the results for all AQs and their SQs.

AQ1: Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to plants?

SQ1.1: Is there evidence of new hazards or risks not previously considered in EFSA scientific opinions?

SQ1.2: Does the new evidence have any implications for EFSA's relevant scientific opinions on NGTs applied to plants?

AQ2: Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to microorganisms?

SQ2.1: Is there evidence for new hazards or risks not previously considered in EFSA scientific opinions?

SQ2.2: Does the new evidence have any implications for EFSA's relevant scientific opinions on NGTs applied to microorganisms?

AQ3: Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to animals?

SQ3.1: Is there evidence for new hazards or risks not previously considered in EFSA scientific opinions?

SQ3.2: Does the new evidence have any implications for EFSA's relevant scientific opinions on NGTs applied to animals?

DATA AND METHODOLOGIES

2

Data

2.1

EFSA was requested to consider any new evidence emerging from the literature search with potential impact on the risk assessment considerations included in past EFSA scientific opinions on plants, microorganisms and animals obtained by NGTs. Table 1 reports the opinions that have been published by EFSA in this field.

Methodologies

2.2

Problem formulation

2.2.1

A detailed description of the problem formulation is reported in the protocol that is included in Annex A. The problem formulation has been made operational by translating the ToRs into AQs, in line with the ‘Guidance on protocol development for EFSA generic scientific assessments’ (EFSA Scientific Committee, 2023).

Literature search

2.2.2

EFSA developed search strings and inclusion/exclusion criteria assisted by a multidisciplinary group of scientists, and ran the searches followed by analysis of the evidence. The experts from the GMO Panel's working groups (WGs) on Molecular Characterisation (MC WG) and the Cross‐Cutting WG (XC WG) were consulted on the search strings, as well as on the scientific publications identified for plants, microorganisms and animals obtained by NGTs, as potentially relevant to the mandate ToRs. More details on the search methodology are included in the protocol in Annex A.

All search strings for AQ1–3, presented in tables 5–13 of the protocol, are constructed from four key terms: New genomic techniques AND Safety AND Species AND Time limits. The NGT terms used cover a variety of expressions that are interchangeably used when mentioning NGTs as defined in Section 1.2, including terms related to specific techniques such as prime editing, base editing, TALEN, ZFN. For the term Species, the terms were based on those used in EFSA GMO Panel (2022a) for plants, EFSA GMO Panel (2024) for microorganisms and Eenennaam (2023) for animals, taking into the account additional terms suggested by public consultation.

The time limits for each of the searches and the dates of searches are presented in Table 2.

Consultation

2.2.3

In line with its policy on openness and transparency, EFSA consulted EU Member States and its stakeholders by an online public consultation. All stakeholders were invited to submit their comments on the draft protocol between May 2025 and June 2025.4 Following this consultation process, the document was revised by EFSA and consulted with the XC WG. The outcome of the online public consultation is reported in Annex which is published on EFSA's website together with the first biannual report (EFSA, 2025) and was taken into account when amending the protocol (Annex A).

ASSESSMENT

3

The number of publications retrieved after each step of the literature search is presented in Table 3. These results are discussed in Section 3.1, ToR1, the criteria for inclusion and exclusion in Section 3.2, ToR2 and the data and evidence in retrieved publications in Section 3.3, ToR3.

ToR1. Develop and validate search strategies to be used for searching of scientific literature databases with adequate sensitivity and specificity to identify studies that could be relevant as regards the safety, risk assessment and other considerations examined by EFSA in its relevant scientific opinions concerning NGTs applied to plants, microorganisms and animals

3.1

Reference publications listed in Table 4 that are relevant for all AQs were used for identifying search terms as well as validating the search strategy.

Overall, the search strategies for all three AQs are of satisfactory sensitivity and specificity since (i) all reference publications were identified and (ii) low number of retrieved publications with no relevance to the topic of NGTs were retrieved. For all AQs, the possible limitations of the search strategies, related to the key terms described in Section 2.2.2, were described in the first report of this mandate (EFSA, 2025) and are still applicable to this report. However, following the outcome of the public consultation, additional keywords were added to the search string to include additional NGTs, terms describing safety and species. The full list of changes is provided in Annex A, Section 4. The search strings could further evolve to include new terms when needed as the horizon scanning methodology is tested and as the scientific knowledge progresses.

AQ1. Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to plants?

3.1.1

The search strings used for AQ1 are the same as those described in the first report for this mandate (EFSA, 2025), with modifications listed in the protocol (Annex A). The modifications include adding keywords suggested by Public Consultation (see Annex A to this report and Annex  B to EFSA, 2025), and adding Pubmed as the source of evidence. In order to assess the impact of modifying the search strings on the number of publications retrieved, the search was run with the time limits set to beginning of 2022, as in the first report. The number of publications retrieved, divided by publication date, is presented in Table 5.

As shown in Table 4, adding additional search strings and Pubmed as the source of evidence led to retrieving studies not covered by the first search. However, after the two levels of screening none of these publications published between 2022 and 2024 were considered relevant for this mandate. Nevertheless, the modifications introduced in this report will be maintained in the next literature searches, to ensure all relevant evidence is retrieved.

AQ2. Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to microorganisms?

3.1.2

The search strings used for AQ2 described in the protocol (Annex A) take into account the suggestions made in Public Consultation (Annex B to EFSA, 2025). During the validation of the search strings, and to ensure harmonised understanding of the search criteria by all reviewers, a calibration activity was performed in which all reviewers screened a random subset of 100 publications. A number of irrelevant publications was identified and various approaches to refine the search strategy were tested. Similarly to the horizon scanning for the GMO Panel's opinion on new developments in biotechnology applied to microorganisms (EFSA GMO Panel, 2024), only primary research publications were retrieved from the databases (see Annex A). The term “NOT biosensors” was also added, to exclude all publications in which CRISPR/Cas systems are used for detection of microorganism, e.g. in environmental, clinical or food samples. Removing publication types such as reviews or book chapters could lead to potentially missing relevant information if its metadata was incorrect. However, as described in Section 3.2.2, the opposite was observed‐ despite this restriction, some reviews, editorials, book chapters, etc., were still retrieved.

After the calibration activity it was tested whether the use of the word “human” in the title or abstract of a publication suggesting medical applications that are out of scope of this mandate, can be linked to the relevance of retrieved publications. The summary of publications divided by use of the term “human” in the search is presented in Table 6.

The majority of publications included after title/abstract screening and all those considered relevant after full‐text screening did not contain the term “human”. The term “human” could be considered when exploring the possibility of artificial intelligence (AI)‐assisted title/abstract screening or prioritisation of publications, but it will not be used to exclude publications a priori, not to compromise the sensitivity of the search.

AQ3. Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to animals?

3.1.3

The search strings used for AQ3 described in the protocol (Annex A) take into account the suggestions made in Public Consultation (Annex  B to EFSA, 2025). During the validation of the search strings, and to ensure harmonised understanding of the search criteria by all reviewers, a calibration activity was performed in which all reviewers screened a random subset of 100 references. A number of irrelevant publications was identified and various approaches to refine the search strategy were explored.

After the calibration activity, it was tested whether the relevance of the retrieved studies for AQ3 correlates to the species mentioned in the title/abstract (see Annex A for a list of species divided into agri‐food and model animals). It was hypothesised that studies focusing on model animals might be more often linked to early‐stage studies (including studies on mammalian cell lines) and/or medical applications and therefore less relevant for the mandate. The summary of publications divided by groups of animals is presented in Table 7.

As presented in the table, the majority of publications included after title/abstract screening described research on agri‐food animals, and the list of publications included after full‐text screening still contained publications on both agri‐food and model animals. This indicates that studies performed on both groups of animals might contain relevant information and the model animals will not be excluded from the search terms in the upcoming reports for this mandate.

ToR2. Develop and pilot criteria for evidence inclusion and exclusion, and critical appraisal tools to support the assessment of relevant published scientific evidence

3.2

Possible limitations of the used inclusion/exclusion criteria were described in the first report for this mandate (EFSA, 2025), and all previous considerations apply to the criteria used for all AQs (see Annex A, Tables 2 and 3). For all three AQs, it was observed that some publications were identified by search strings designed for one of the other groups of organisms, e.g. a publication potentially relevant for microorganisms was retrieved only by a search string for animals. Such publications were transferred to the more appropriate reference pool and screened again by two reviewers for their relevance. The data presented in Table 3 shows the amount of references after this re‐assignment. Table 8 shows the number of publications transferred between respective groups.

Overall, none of the publications re‐assigned to another group of organisms were considered relevant after full‐text screening, confirming that the applied search strings per organism group is sufficiently sensitive and specific. However, certain publications might contain keywords related not to the organism in which the genetic modification was performed (which might not even be explicitly stated), but contain keywords related to another organism – e.g. a bacterial Cas was used to modify a mammalian gene (the species not specified in abstract), or a plant microbial pathogen was modified (the term “pathogen” being used, which is not a search term for microorganisms per se). Such re‐assignments can be performed as needed and do not impact the sensitivity of the search.

AQ1. Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to plants?

3.2.1

The criteria for evidence inclusion and exclusion used for AQ1 are the same as reported in the first report for this mandate (EFSA, 2025) and all previous considerations apply. The protocol (Annex A) has been amended to improve clarity of the section describing these criteria.

AQ2. Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to microorganisms?

3.2.2

The outcome of the pilot confirms that the search and screening strategies using the developed inclusion/exclusion criteria both are specific and sensitive and are fit for purpose in addressing the mandate. To ensure alignment among reviewers on the use of the inclusion/exclusion criteria, a calibration exercise was performed using a random subset of 100 publications reviewed by all. Any differences in the assessment were discussed and resolved before proceeding with the full set of publications. During the title and abstract screening a conservative approach was adopted; if it was unclear whether a study met all inclusion criteria, it was marked as ‘unclear’ and followed by full‐text screening. In addition, the publications for which an abstract was not available were also marked as ‘unclear’ and followed by full‐text screening.

The studies retrieved by the literature searches but eventually excluded could be categorised as:

• Not primary research studies; even though the search strings were designed to eliminate these studies based on their metadata, this strategy did not eliminate all reviews, opinions, editorials, etc. This is because not all publications have complete or correct metadata. Such publications were mostly excluded at the title/abstract level. Criterion for exclusion: publication type
• Studies focusing on regulatory and societal aspects of NGTs, market value. Criterion for exclusion: study design
• NGTs for applications outside of the remit of EFSA, e.g. to develop biosensors for detection of food contaminants (although this is partly resolved already at the level of search strings which exclude the term “biosensor*”), medicinal uses, e.g. vaccine development, except when the abstract suggested potential side effects of the technique itself. Criterion for exclusion: Scope/intended use
• Studies of the intrinsic bacterial CRISPR mechanism and not the use of a technique to modify the microorganisms to obtain new traits. Criterion for exclusion: Technique
• Studies on other organisms. Criterion for exclusion: Organisms

After the second step of screening, 29/35 studies (82.9%) were excluded due to the criterion: Outcome/conclusion. In these cases, the abstract suggested some possible new evidence whose significance could only be assessed after full‐text screening and critical appraisal. These studies were mostly:

• Functional gene studies in which gene editing was used as a tool, e.g. to knock out a gene.
• Studies characterising a new nuclease, prime editor, base editor, most often with improved properties, e.g. superior efficiency or increased specificity.
• Studies reporting on applying gene editing for the first time in a given microorganism.
• Novel methods or protocols to improve gene editing efficiency, e.g. by modifying reaction temperature or salt concentration.
• Bioinformatic algorithms for off‐target prediction and/or sgRNA design.

Moreover, compared to the screening of publications for AQ1 and AQ3 (Sections 3.2.1 and 3.2.3), a higher number of publications (19/35, 54.3%) were excluded due to the criterion: Scope/intended use. Such studies were mostly proof‐of‐concept, and their aim was not to generate any product that could require an EFSA risk assessment, but to test the efficacy or specificity of an NGT for a specific species. These studies were screened in full text because based on their abstract it was not clear what the possible application could be.

AQ3. Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to animals?

3.2.3

The outcome of the pilot confirms that the search and screening strategies using the developed inclusion/exclusion criteria both are specific and sensitive and are fit for purpose in addressing the mandate. To ensure alignment among reviewers on the use of the inclusion/exclusion criteria, a calibration exercise was performed using a random subset of 100 publications reviewed by all. Any differences in the assessment were discussed and resolved before proceeding with the full set of publications. During the title and abstract screening, a conservative approach was adopted; if it was unclear whether a study met all inclusion criteria, it was marked as ‘unclear’ and followed by full‐text screening. In addition, the studies for which abstract was not available were also marked as ‘unclear’ and followed by full‐text screening.

The studies retrieved by the literature searches but eventually excluded could be categorised as:

• Not original studies; mostly excluded at the title/abstract level. Criterion for exclusion: publication type
• Studies focusing on regulatory and societal aspects of NGTs, market value. Criterion for exclusion: study design
• NGTs for uses other than agri‐food, e.g. to develop vaccines, or animals were used for human disease studies, except when the abstract suggested potential side effects of the technique itself. Criterion for exclusion: Scope/intended use
• Studies on other organisms. Criterion for exclusion: Organisms

After the second step of screening, 26/30 studies (86.7%) were excluded due to the criterion: Outcome/conclusion. In these cases, the abstract suggested some possible new evidence whose significance could only be assessed after full‐text screening and critical appraisal. These studies were mostly:

• Functional gene studies in which gene editing was used as a tool, e.g. to knock out a gene.
• Studies characterising a new nuclease, prime editor, base editor, most often with improved properties, e.g. superior efficiency or increased specificity.
• Studies reporting on applying gene editing for the first time in a given organism.
• Novel methods or protocols to improve gene editing efficiency, e.g. by modifying reaction temperature or salt concentration.
• Bioinformatic algorithms for off‐target prediction and/or sgRNA design.

ToR3. Extract and summarise relevant data and evidence and assess whether any new data and evidence may have implications for EFSA relevant scientific opinions

3.3

AQ1. Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to plants?

3.3.1

In this horizon scan of NGT plants, while most studies were excluded based on exclusion criteria (Table 3), five publications were critically discussed further with experts to assess whether they provide potentially relevant evidence which may have an impact on the previous EFSA conclusions (Table 1). These studies are reported in Appendix A and regard the development of a new large scale genome editing technique (Sun et al., 2025), the characterisation of off‐target rates (Seol et al., 2025), advances in in silico prediction tools (Mekonnen et al., 2025) and the application of genome editing in organelles (Mangu et al., 2025) and promoter regions (Ke et al., 2025).

Sun et al. (2025) describe the proof‐of‐concept application of programmable chromosome engineering (PCE) and scarless programmable chromosome engineering using Re‐pegRNA (RePCE) which enable scarless chromosomal insertions, deletions, inversions, replacements and translocations at megabase (Mbp) scale. The authors additionally describe an AI‐assisted tool to generate highly efficient Cre recombinase and Lox sites variants that can be used for CRE‐Lox excisions of DNA fragments. EFSA has already indicated in its opinions that a plant with stable integration of the CRISPR/Cas system or where any residual transgene is present is to be considered transgenic, and its risk assessment must follow all provisions laid down in current EU GMO regulations (EFSA GMO Panel, 2020; EFSA GMO Panel, 2022a). Taking this into consideration, the strategies of scarless programmable chromosome engineering, proposed in this publication, could be relevant to produce non‐transgenic NGT plants. It must be also noted that large‐scale chromosomal rearrangements such as the ones described in Sun et al. (2025) can also be found in nature and have been exploited by plant breeders and EFSA previous opinions commented on the possibility of large chromosomal rearrangements.

Two publications focused on the number of off‐target changes and their detection. Seol et al. (2025) employed CRISPR/Cas9 to disrupt genes associated with male sterility in tomato. Off‐target mutations were initially predicted using Cas‐OFFinder (Bae et al., 2014) and subsequently validated by sequencing. The analysis revealed low but variable off‐target frequencies among independently transformed lines. The observed variability in off‐target mutations was attributed to differences in editing specificity. Although not further investigated in this study, these differences in off‐target mutations may be due to the positional effect of the Cas9 cassette insertion site and the variability in its expression level. However, in agreement with EFSA's work (EFSA GMO Panel, 2020), the off‐target mutations are of the same type as those genetic changes observed in conventional breeding or those generated by random mutagenesis techniques. Although in silico prediction tools greatly facilitate the design of gRNAs, they rely on the availability of reference genomes which may differ from the genome of the specific cultivar or line modified. To address this limitation, Mekonnen et al. (2025) developed a variant‐aware Cas‐OFFinder tool which incorporates SNPs and small indels to improve prediction accuracy, though structural variants are not yet incorporated in the tool. Despite its utility for gRNA design, experimental validation remains essential to confirm off‐target events predicted by in silico tools.

Mangu et al. (2025) described chloroplast editing using transcription activator‐like effector nuclease (TALE)‐cytidine deaminase base editing. While this approach successfully knocked out the target gene, it also introduced a consistent off‐target mutation. This observation suggests that the frequency and nature of off‐targets in the plastid genome may differ from those in the nuclear genome, highlighting the need for further investigation of off‐target activity in organellar genomes.

These knockouts were complemented by classical transgenesis, where the mutated gene was inserted into the nucleus and the gene product (resulting protein) redirected to the chloroplast by a transit peptide to restore the phenotype. The functional complementation of mutants through nuclear expression of chloroplastidial genes and their subsequent relocation to the organelle is not new. Such approaches can be classified as cisgenesis or intragenesis, for which risk assessment considerations have already been discussed by EFSA EFSA GMO Panel (2012b, 2022c).

Ke et al. (2025) used CRISPR/Cas9 to knock in stress‐responsive cis‐regulatory elements (CREs) into promoters of Arabidopsis thaliana genes, generating heritable, transgene‐free lines with fine‐tuned regulation of downstream targets without altering open reading frames. Because the CREs are derived from the breeder's gene pool, the plants could be considered cisgenic. These types of modifications do not introduce novel hazards compared to conventional breeding, as previously stated by EFSA (EFSA GMO Panel, 2012b, 2022a). Genome‐wide studies show that CREs are dynamic, with single nucleotide polymorphisms (SNPs), indels and transposable element (TE) insertions reshaping regulatory networks (O'Malley et al., 2016). TE‐derived CREs are also frequently repositioned across lineages, demonstrating natural turnover and reshuffling (Du et al., 2024). Importantly, like every sequence in the genome, cis‐regulatory elements are dynamic genomic features that can change over time through the accumulation of spontaneous mutations and the activity of TEs for example. Such changes have already been selected through classical breeding practices or during domestication of crop plants.

None of the studies discussed provided new evidence that changes the previous EFSA conclusions.

AQ2. Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to microorganisms?

3.3.2

In this literature horizon scan of NGT microorganisms, while most studies were excluded based on exclusion criteria (Table 3), 10 publications were discussed further with experts to assess whether they provide potentially relevant evidence, which may have an impact on the previous EFSA conclusions and recommendations (Table 1). These studies are reported in Appendix A and concern: the environmental risk assessment (Chang, 2024), containment and kill switches (Asin‐Garcia et al., 2024; Hartig et al., 2024; Lamb et al., 2025); horizontal gene transfer (HGT) potential of chlorine‐resistant genes (Huang et al., 2025); CRISPR/Cas editing leading to unintended chromosomal translocations (Hou et al., 2025), deletions (Ogata et al., 2025) and transpositions (Lee & Kim, 2025); high number of heterokaryons (Virgílio et al., 2025) or loss of heterozygosity (Ramírez‐Zavala et al., 2024) as a potential consequence of gene editing.

Chang (2024) describes the creation of large chromosomal segment deletions of gene clusters responsible for the production of aflatoxin, cyclopiazonic acid and ustiloxin B in Aspergillus flavus by a dual CRISPR/Cas9 system. The author claims that A. flavus strains possessing defective aflatoxin gene clusters could be used to manage preharvest aflatoxin contamination of crops as biological control. The author underlines that, since no foreign DNA is integrated into genomes, approval of applications for experimental use permits by the US Environmental Protection Agency or similar regulatory agencies in other countries would be easier. The author also discusses the potential of off‐target mutations.

EFSA is aware of the possibility of generation of off‐target mutations in fungi by using NGTs or other techniques and in its scientific opinions clearly states the need to investigate intended modification(s) as well as potential off‐target mutations by, whole genome sequencing (WGS) of the gene‐edited strain as compared to the parental strains (e.g. EFSA GMO Panel, 2024; EFSA Scientific Committee et al., 2025). In addition, the WGS analysis should be supported by phenotypic data to study whether potential off‐target mutations could result in adverse phenotypic traits (e.g. EFSA Scientific Committee, 2025). Therefore, this study does not present any information that would change the recommendations and conclusions of the existing EFSA opinions.

Asin‐Garcia et al. (2024) describe the GenoMine, a CRISPR/Cas9‐based kill switch for biocontainment of Pseudomonas putida. This study shows that NGTs can be used to develop kill switches in bacteria for biocontainment; however, these are not yet effective. This lack of effectiveness in not NGT‐related but is known to occur for containment strategies using other techniques. Lamb et al. (2025) describe the secure design of kill switches in Saccharomyces cerevisiae for biocontainment and discuss the possible escape mechanisms. The authors state that the use of multiple kill switches obtained by NGTs can result in a more effective biocontainment strategy in yeasts. Escape from biocontainment can take place due to off‐target mutations; however, this is not unexpected and is not related to the technique used for biocontainment. Hartig et al. (2024) describe the influence of the environmental conditions on the escape rate of bio‐contained genetically modified microorganisms (GMMs). The authors develop a CRISPR‐based kill switch for E. coli triggered by anhydrotetracycline and evaluate the escape rate in different environmental conditions. The authors discuss the need to consider biotic and abiotic factors that may influence undesirable persistence of the microbe in the environment.

Overall, these three can be considered proof‐of concept studies; the potential products are not expected to enter the EU market in the near future.

The risk assessment considerations for these containment strategies have already been addressed in the opinion on microorganisms obtained through synthetic biology (EFSA Scientific Committee, 2020) where it is stated that such strategies have potential for gene containment, but their efficacy should be assessed on a case‐by‐case basis and be supported by experimental data. Where experimental data are needed for GMMs, these data should be obtained under conditions that reflect as much as possible the natural conditions (biotic, abiotic) of the receiving environment(s) in which the GMM would be introduced (EFSA Scientific Committee, 2025).

Huang et al. (2025) describe in vitro HGT of a newly introduced gene in a synthetic E. coli strain to other microorganisms, under conditions of selective pressure. The observed HGT is not linked to the fact that NGTs were used to introduce the gene in E. coli, but to the potential of HGT irrespective of the method used to introduce the gene. The results are not unexpected since HGT between microorganisms is known to occur but is considered only to result in potential adverse effects under selective environmental pressure, leading to selective advantage of recipient microorganism. Potential adverse effect resulting from HGT is considered in EFSA opinions and needs to be assessed on a case‐by‐case basis (e.g. EFSA GMO Panel, 2024; EFSA Scientific Committee, 2020, 2025). Therefore, this study does not present any information that would change the recommendations and conclusions of the existing EFSA opinions.

Hou et al. (2025) describe the assessment and mitigation of CRISPR/Cas9‐induced non‐targeted translocations. The authors identify these as a risk, develop a trans‐acting reporter system in E. coli to detect the non‐targeted translocations and suggest mitigating strategies. The unintended large‐scale genomic rearrangements are not unexpected and therefore not considered a new hazard. Large rearrangements can be elucidated by making use of long‐read sequencing technologies of WGS. Ogata et al. (2025) describe the CRISPR/Cas9 genome editing of the allodiploid yeast Zygosaccharomyces sp. The authors discuss the fact that in some genome‐edited strains a significant region of one sub‐genome chromosome is missing. The authors also state that the deletion was observed only by polymerase chain reaction (PCR) experiments, and that in the future WGS using short reads, as well as pulsed‐field gel electrophoresis would be necessary to confirm these findings. Lee and Kim et al. (2025) describe the transposition of IS1 in Edwardsiella piscicida developed with the use of CRISPR/Cas9 and lamda‐red recombineering. The authors propose that observed IS1 transposition in the mutants is based on the creation of the double‐stranded break induced by the CRISPR/Cas9 system and not to an off‐target effect. Transposition of IS elements is a common phenomenon in microorganisms that can also occur naturally and is not specific to the use of NGTs.

The requirement to use WGS to molecularly characterise the microorganisms was already reported in the EFSA Opinion (2024). In addition, the suggestion to apply it to other microorganisms is described in the new guidance on the characterisation of microorganisms in support of the risk assessment of products used in the food chain (EFSA Scientific Committee et al., 2025) and is supported by the WGS Statement of EFSA (2024). Therefore, this study does not provide any information on new hazards that have not been addressed by EFSA or would change the recommendations and conclusions.

Virgílio et al. (2025) describe the CRISPR/Cas9‐mediated engineering of the Metarhizium anisopliae filamentous fungus. The study discusses the challenges of the Cas9 integration and reports a large number of heterokaryons observed in the edited strain as compared to the wild type strain. This phenomenon is not linked to the safety of the editing technique but rather to the efficacy of the transformation and is known to occur, similarly to mosaicism in plants/animals. To eliminate this problem, multiple rounds of selection are needed. The study does not present any information that would change the recommendations and conclusions of the previous EFSA opinions.

Ramírez‐Zavala et al. (2024) describe the transformation of Candida albicans using CRISPR/Cas9 system. The authors discuss the fact that loss of heterozygosity (LOH) on the target chromosome is observed when using CRISPR/Cas9 for the generation of C. albicans gene deletion mutants and suggest to always check the mutants for LOH at the target locus, in particular when Cas9‐mediated chromosome breakage is involved. The loss of heterozygosity or structural variations may be a ‘side effect’ of the editing technology. It is not considered a safety issue, but rather a method efficiency one. Moreover, it is not specific to the NGTs, but can also happen as a result of EGTs. Therefore, the study does not present any information that would change the recommendations and conclusions of the previous EFSA opinion.

The EFSA Guidance (EFSA Scientific Committee, 2025) requests that, for GM microorganisms, WGS, as well as information on the stability of the introduced trait (phenotype), is provided. Genetic stability of the introduced trait may also be requested on a case‐by‐case basis.

None of the studies discussed provided new evidence that changes the previous EFSA conclusions.

AQ3. Is there new evidence emerging from the identified studies that may have implications for relevant EFSA scientific opinions on NGTs applied to animals?

3.3.3

Most of the publications identified in this literature horizon scan for NGT animals were excluded based on exclusion criteria (Table 3), whereas eight studies were discussed further with experts to assess whether the results reported therein potentially provide evidence that could change previous EFSA conclusions (Table 1).

Three of these studies address the application of NGT to pigs, and discuss potential adverse effects due to multiplexing (Huai et al., 2025; Peng et al., 2025) and unintended mutations (Huo et al., 2025). Three studies (Chen et al., 2025; Kim et al., 2025; Sin et al., 2025) report potential toxic effects of CRISPR/Cas technology in mice, pigs and non‐human primates, respectively. One publication reports deleterious effects when CRISPR reagents were expressed at high doses in the mitochondrial genome (Barrera‐Paez et al., 2025). Finally, Suvá et al. (2025) reports the early death of two gene‐edited calves with a myostatin (mstn) gene knock out. As further detailed below, no new hazards or risks directly associated with the methodological application of NGTs to animals for food, feed or other agricultural uses have been identified in these studies that would alter the previous EFSA conclusions.

Huai et al. (2025) describe the knock‐out of three genes (GGTA1, CMAH and β4GalNT2) combined with the expression of regulatory transgenes for human complement and coagulation (hCD55, hTM and hEPCR) in pigs with the final aim of reducing the likelihood of xenograft rejection in humans. The authors reported increased susceptibility to infection in the modified pigs, termed 6GE pigs. The three genes knocked out are responsible for producing carbohydrate (sugar) antigens on pig cells, and the increased susceptibility to infection in 6GE pigs is known to be linked to the knock‐out of the sugar‐related genes, which affects innate immunity for instance by means of modification of pathogen recognition or change in microbiota interactions. The knock‐out of the three genes, combined with the expression of human immune and coagulation regulators transgenes (hCD55, hTM and hEPCR), may further disrupt immune balance, increasing vulnerability to pathogens, particularly in conventional, non–pathogen‐free environments. The increased disease susceptibility is therefore not linked to the use of CRISPR/Cas9 technology to knock out the genes, but to the nature of the genes that were inactivated.

The same 6GE pigs were studied by Peng et al. (2025), who described physiological health status and organ function in the modified animals. Apart from increased susceptibility to infection, the authors also reported reduced weight and lower reproductive performance in 6GE pigs compared to the non‐modified controls. These results are understandable considering the knocked out genes CMAH, GGTA1 and β4GalNT2 are also expressed in the surface of cells within the testes, which can lead to changes in cell‐to‐cell interactions. Thus, once more, the observed effects are very likely due to the genes being knocked out and not to the technique itself. It must be noted that somatic cell nuclear transfer (SCNT), used in the two studies as part of the breeding process, has been known to have drawbacks (e.g. large offspring, high perinatal mortality and fertility issues) which are mostly observed in the founder animals and not in the later generations that will be marketed. These issues caused by SCNT are not NGT‐specific, as this technique can also be used, e.g. for transgenesis or cloning. The possibility of altered susceptibility to pathogens and infections, as well as the potential concerns for animal welfare, were already considered in the GMA‐NGT scientific opinion (EFSA GMO Panel, 2025); therefore, these publications do not provide any evidence pointing to a potentially new safety concern. Additionally, Peng et al. (2025) reported random integration of human transgenes into the pig genome, associated to the use of EGTs in addition to NGTs in this study. Random integrations of transgenes by EGTs a known phenomenon.

The generation of MSTN‐edited calves by means of CRISPR/Cas9 and SCNT is described in Suvá et al. (2025). A simple protocol to efficiently generate MSTN‐KO calves without using DNA templates was developed by combining CRISPR/Cas9 RNP knock‐out of the MSTN gene with SCNT. Cell samples from ‘prize‐winning’ animals were used in the study as a proof of concept that gene editing can be applied in breeding programs offering an alternative to traditional crossbreeding. Only one MSTN‐KO calf was generated in the first generation, and it died early on due to respiratory disease. The only second‐generation MSTN‐KO calf obtained from cells of the first‐generation gene‐edited calf also had an early death due to extreme heat. These deaths could be a consequence of the MSTN‐KO phenotype, which has been reported to lead to higher sensitivity to respiratory diseases and higher sensitivity to heat in cattle (Aiello et al., 2018). However, this is not mentioned in the publication. On the other hand, the birthing rate of cloned calves is known to be low, and death of cloned calves is common. Death of the two calves presented in this work, attributed to two different causes, is therefore highly likely to be associated with either unforeseen factors or to the SCNT technique, and not to CRISPR/Cas9 technology per se. As discussed before (Peng et al., 2025), SCNT and transfection might lead to accumulation of epigenetic changes and reduced embryo survival.

Huo et al. (2025) studied whether mutations in GGTA1‐knockout pigs are transmitted to the following generations. The authors reported unexpected random integration of the sgRNA‐Cas9 plasmid into the pig's genome, something that has been previously reported in cattle. Unintended de novo mutations were also observed, but they are likely associated to cell stimulation for plasmid introduction and thus would not be off‐target effects of CRISPR/Cas9. No evidence of new hazards is provided that would change previous EFSA conclusions.

Chen et al. (2025) used CRISPR/Cas9 to reprogram metabolic pathways in a mouse model for the human disease hereditary tyrosinaemia type I. It was noted that the mice used in this study were shown to be prone to liver tumours, including control animals; therefore, the observed tumorigenesis cannot be specifically linked to the use of CRISPR/Cas9. In addition, an adeno‐associated viral (AAV) vector was used for gene delivery. AAV enters the cell easily but it is known to be able to integrate into the host genome on an occasional basis. The combination of CRIPSR/Cas9 with AAV could increase the number of undesired integrations. However, this is known and thus provides no evidence of new concerns that would change previous EFSA conclusions.

Kim et al. (2025) describe the generation of a CRISPR/Cas9 pig that expresses constitutively the protein Cas9, with the final aim of supporting faster generation of gene‐edited pigs for different purposes. Similarly to other studies previously discussed, the authors observed adverse effects potentially caused by the use of SCNT and toxicity of Cas9, such as embryo lethality, high neonatal mortality and DNA damage, among others. Some of the issues reported seem to be associated to the use of SCNT; however, limitations of this study (e.g. small sample size studied and a low level of detail provided in the publication) complicate the interpretation of the results. The cellular toxicity of Cas9 is due to its constitutive and ubiquitous high‐level expression and it is a known phenomenon, also observed in microorganisms, or in plants, where high expression levels of Cas9 has also been reported to potentially lead to more DNA breaks and off‐targets.

Sin et al. (2025) studied the efficacy and safety of a genome editing system based on CRISPR/Cas9 and AAV vectors to treat choroidal neovascularization in non‐human primates. The study reports deleterious effects by the use of AAV8‐Cas9 with or without co‐transformation with sgRNA. The fact that the effects were observed irrespective of the sgRNA indicated that they are not caused by the CRISPR/Cas9 system itself. These effects are most probably due to very high dosage of the Cas9 protein, and have also been shown for other exogenous proteins.

Barrera‐Paez et al. (2025) describes the efficiency and safety of a mitochondrial DddA‐derived cytosine base editor (DdCBE) to edit the mitochondrial DNA in mice. DdCBE was packaged in recombinant AAV9 and intravenously administered by retro‐orbital injections into mice. The study reports DdCBE induced off‐target edits that resulted in severe adverse effects in mice at the highest AAV9‐DdCBE dose tested, but not at lower doses. These data indicate that the adverse effects of DdCBE are not related to the use of the base editor itself, but to toxicity of the high dose used. It is known that the use of base editors, can lead to off‐target effects and that the probability of off‐target effects increases with the dosage (EFSA GMO Panel, 2025).

It is worth noting that the majority of the adverse effects linked to the expression of Cas9 protein are observed in gene therapy applications, or in animals in pre‐commercial stages and are not expected to be present in animals potentially ready for the market, which fall under the remit of EFSA's risk assessment. As mentioned in the EFSA GMO Panel (2025) scientific opinion, pre‐commercial stages need to be considered when they provide important information for the risk assessment; nevertheless, ethics and socio‐economic aspects related to animal welfare associated with gene editing technology are out of the scope of EFSA. Side effects due to high expression levels of Cas protein or Cas‐gRNA complex, in terms of increased off‐target effects or cellular toxicity, are well known and were already considered in the EFSA opinion on SDNs (EFSA GMO Panel, 2020) and in other publications (Álvarez et al., 2022; Cho et al., 2018; Zhang & Voigt, 2018). All the findings report the need to reduce expression levels of Cas protein, to engineer Cas9 variants or to develop tissue‐specific expression systems, in order to reduce toxicity.

None of the studies discussed provided new evidence that changes the previous EFSA conclusions.

CONCLUSIONS

4

The outcome of the pilot screening for AQ2 and AQ3 demonstrates that the search strategy (ToR1) is both sensitive and specific. It reliably identifies publications of relevance while limiting the inclusion of irrelevant publications.

The outcome of the pilot screening for AQ2 and AQ3 also confirms that the inclusion and exclusion criteria (ToR2) are specific and appropriate for the mandate. The application of a conservative approach to studies of uncertain relevance provides additional assurance that no significant publications were overlooked during the initial screening phase.

In line with the EFSA protocol and the AQs, EFSA concluded that none of the studies identified through the literature search introduces hazards or risks not previously considered in EFSA scientific opinions (ToR3).

RECOMMENDATIONS FOR EFSA'S FUTURE WORK

5

EFSA will continue to systematically assess the suitability of the search strings used in the literature screening protocol as NGTs evolve and their applications in the agri‐food and environmental domain relevant to the past EFSA opinions expand. Any modifications to the protocol will be documented and justified. EFSA will also ensure consistency in the screening process through internal calibration exercises between reviewers.

In preparation for the two scientific reports for this mandate, more than 1300 studies were screened at title/abstract level for each AQ. These screened studies could serve as a training dataset for AI‐assisted screening. DistillerSR includes functionality that enables AI to act as a second reviewer5 during the title/abstract screening stage (Cagnoni et al., 2023). Should this be successfully tested and implemented, such change in methodology will be reported in one of the future biannual reports. Despite the high number of screened studies, the number of relevant publications identified at full‐text level remains limited, and these studies employ diverse experimental designs. Therefore, the development of structured critical‐appraisal tools is currently considered to offer limited added value. EFSA will continue to follow a qualitative approach for evidence extraction and appraisal.

As the body of assessed evidence expands over future reporting cycles, EFSA may maintain a consolidated list of reviewed publications in a dedicated PowerBI report. PowerBI would facilitate filtering and categorisation of studies – for example by technique used, species or thematic tags such as bioinformatic analyses, machine learning applications, or functional gene studies. Over time, this could support the development of a comprehensive repository of publications relevant to NGTs for food, feed and environmental risk assessment, providing an important resource for future guidance development and related EFSA activities.

ABBREVIATIONSAAVadeno‐associated virusAIartificial intelligenceAQassessment questionCBEcytosine base editorCRECis‐regulatory elementCRISPRclustered regularly interspaced short palindromic repeatsDdCBEDddA‐derived cytosine base editorEGTestablished genomic techniqueGMMgenetically modified microorganismGMOgenetically modified organismHGThorizontal gene transferIRinverted repeatISinsertion sequenceLOHloss of heterozygosityMCmolecular characterisationNGTnew genomic techniquePCEprogrammable chromosome engineeringPCRpolymerase chain reactionRePCEscarless programmable chromosome engineering using Re‐pegRNASCNTsomatic cell nuclear transferSNPsingle nucleotide polymorphismSQsub‐questionTALEtranscription activator‐like effector nucleasesTEtransposable elementToRTerms of referenceWGworking groupWGSwhole genome sequencingXCcross‐cutting

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2025‐00504

COPYRIGHT FOR NON‐EFSA CONTENT

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Supporting information

Annex A: Protocol for ‘Request to provide scientific and technical assistance for a regular horizon scanning to assess new scientific data on plants, animals, microorganisms and products thereof obtained by new genomic techniques’.

Annex B: Publications excluded after full‐text screening.