Sunday Evening News 371 - Week 15 - 2024
Weekly report on genetic engineering, genome editing, biotechnology and legal regulations.
April 2024-04-08 - 2024-04-14
Meetings – Conferences / Veranstaltungen - Konferenzen
EU-Parlament: 24.04.2024: 1. Lesung und Abstimmung zum Kommissionsvorschlag genomeditierte Pflanzen (S. 8)
EU Parliament: 24 April 2024: 1st reading and vote on Commission proposal on genome-edited plants (p. 8)
https://www.europarl.europa.eu/doceo/document/OJ-9-2024-04-22_DE.pdf
Press Releases -Media / Presse- und Medienberichte
European Committee of the Regions: EU rules on new genomic techniques and on plant reproductive material will put the
future of farmers and consumers at risk, local leaders warn
https://cor.europa.eu/it/news/Pages/21-nat-meeting.aspx
09.04-2024 ENVI-Ausschuss-Sitzung Aussprache zum ANSES-Report - ENVI Committee meeting Debate on the
ANSES report:
Unterlagen: ANDES-Report: https://www.europarl.europa.eu/meetdocs/2014_2019/plmrep/COMMITTEES/ENVI/DV/2024/04-09/BIORISK2021SA0019_EN.pdf
Präsentation-ANSES-Schuler:
Informationsdienst Gentechnik: EU-Kommission weist französische Kritik am Gentechnik-Entwurf zurück
https://www.keine-gentechnik.de/nachricht/34929?cHash=458ea18f3a6b941ba1596ad66d903e29
Struna H.: EU-Kommission verteidigt Vorschlag zu Gen-Pflanzen gegen französische Einwände
EU Commission defends proposal on gene-edited plants against French food safety agency
DRAFT MINUTES COUNCIL OF THE EUROPEAN UNION, (Agriculture and Fisheries) 26 March 2024
https://data.consilium.europa.eu/doc/document/ST-8350-2024-INIT/en/pdf
SAG: Marktüberwachung in der EU: Erneut Antibiotikaresistenzgene aus GVO in Lebensmittelenzymen entdeckt
Only some selected press releases or media reports are listed here. The daily up-date of the press releases and
media reports are ►here: April week 15
Publications – Publikationen
DFG: Ständige Senatskommission zur gesundheitlichen Bewertung von Lebensmitteln (SKLM) Lebensmittel- und
Ernährungsforschung in Deutschland
Atimango A.O, Wesana J., Kalule S. W., Verbeke W., De Steur H. (2024): Genome editing in food and agriculture: from
regulations to consumer perspectives Current Opinion in Biotechnology 87, 103127| https://doi.org/10.1016/j.copbio.2024.103127
Genome editing (GE) has emerged as a technology that could revolutionize food and agricultural production. While its advent has evoked enthusiasm for a more sustainable food system, there exists heterogeneity in regulations and public opinions regarding the technology. This review discusses evidence on the implications of government regulations on GE, and perceptions of genome-edited (GEd) food and related regulations. The review highlights consumers’ positive attitude and preference for GEd foods when compared with genetically modified foods, despite the limited awareness and knowledge of GE technology. While policy changes might trigger debates, providing tailored benefits, information to consumers could further improve their attitude toward GE.
https://www.sciencedirect.com/science/article/abs/pii/S0958166924000636?dgcid=rss_sd_all
Buchman L. W, Goldsmith C. L., Heitman E., Kang K.E., Liu X. (2024): Public trust in regulatory agencies and support for
policies on agricultural gene drive. RPR | https://doi.org/10.1111/ropr.12610
Public trust in government agencies plays an important role in the formation of public opinion about public policy issues. However, the association between public trust in regulatory agencies and public support for policy development in emergent biotechnologies such as gene drive is not well understood. The United States Department of Agriculture (USDA), the Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA) are tasked with coordinating and regulating biotechnology. Drawing on past literature, this study examines how public trust in these federal agencies is associated with public opinion on various options for gene drive policy. Using data from a nationally representative public opinion survey (n = 1220) conducted in 2021, our statistical analyses show that respondents who report higher levels of trust in regulatory agencies are more likely to support policy proposals that promote gene drive research and ultimately, may lead to regulatory policies that allow gene drive to be researched and employed to manage agricultural pests, establishing a pathway for scientists, developers, producers, and consumers alike to realize the benefits of this technology.
https://onlinelibrary.wiley.com/doi/10.1111/ropr.12610?af=R
Katayama S., Watanabe M., Kato Y., Nomura W., Yamamoto T. (2024): Engineering of Zinc Finger Nucleases Through Structural
Modeling Improves Genome Editing Efficiency in Cells. Adv. Sci., 2310255. https://doi.org/10.1002/advs.202310255
Genome Editing is widely used in biomedical research and medicine. Zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector (TALE) nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFN-encoding DNAs can be easily packaged into a viral vector with limited cargo space, such as adeno-associated virus (AAV) vectors, for in vivo and clinical applications. ZFNs have great potential for translational research and clinical use. However, constructing functional ZFNs and improving their genome editing efficiency is extremely difficult. Here, the efficient construction of functional ZFNs and the improvement of their genome editing efficiency using AlphaFold, Coot, and Rosetta are described. Plasmids encoding ZFNs consisting of six fingers using publicly available zinc-finger resources are assembled. Two functional ZFNs from the ten ZFNs tested are successfully obtained. Furthermore, the engineering of ZFNs using AlphaFold, Coot, or Rosetta increases the efficiency of genome editing by 5%, demonstrating the effectiveness of engineering ZFNs based on structural modeling.
https://onlinelibrary.wiley.com/doi/10.1002/advs.202310255
Lee, S., Park, S.H., Jeong Jeong, Y. et al. (2024): Optimization of CRISPR/Cas9 ribonucleoprotein delivery into cabbage
protoplasts for efficient DNA-free gene editing. Plant Biotechnol Rep | https://doi.org/10.1007/s11816-024-00901-9
The CRISPR/Cas9-based gene editing system for the direct delivery of pre-assembled Cas9 ribonucleoproteins (RNPs), consisting of a Cas9 nuclease and a single guide RNA (sgRNA), into plant protoplasts enables DNA-free gene editing without introducing foreign gene into plants. Here, we described the optimization of CRISPR/Cas9 RNPs delivery into cabbage protoplasts for efficient DNA-free gene editing. We determined the insertion and deletion (indel) frequency of BoMYBL2-1, a negative regulatory gene for anthocyanin biosynthesis in cabbage (Brassica oleracea var. capitata). We optimized the molar ratio of Cas9 to sgRNA and the incubation time of RNP–protoplast transfection to enhance the indel frequency under various conditions. Based on the BoMYBL2-1 nucleotide sequences, we designed nine sgRNAs to target BoMYBL2-1. Our in vitro digestion assay showed that all sgRNAs were able to cleave the targeted fragment. When the sgRNA and Cas9 proteins were subsequently transfected into protoplasts isolated from cabbage cotyledons, the deep sequencing results showed that the indel frequency of sgRNAs in BoMYBL2-1 was the highest (7.4%) with sgRNA3. We compared various molar ratios of Cas9 and sgRNA and incubation times of RNP–protoplast transfection to optimize transfection and ensure high indel frequency. The highest frequency was observed when the Cas9:sgRNA ratio was 1:10. Furthermore, when the incubation time for RNP–protoplast transfection was 1 min and 3 min, the indel frequency was higher than 25%. Altogether, these results provide valuable information on the optimized conditions for high-efficiency gene editing using CRISPR/Cas9 RNP delivery into cabbage protoplasts
https://link.springer.com/article/10.1007/s11816-024-00901-9
Tadesse D., Estella F., Yee E.F., Wolabu T. W. et al. (2024): Sorghum SbGhd7 is a major regulator of floral transition and
directly represses genes crucial for flowering activation. New Phytologist | https://doi.org/10.1111/nph.19591
Molecular genetic understanding of flowering time regulation is crucial for sorghum development. GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (SbGhd7) is one of the six classical loci conferring photoperiod sensitivity of sorghum flowering. However, its functions remain poorly studied.
The molecular functions of SbGhd7 were characterized. The gene regulatory network controlled by SbGhd7 was constructed and validated. The biological roles of SbGhd7 and its major targets were studied.
SbGhd7 overexpression (OE) completely prevented sorghum flowering. Additionally, we show that SbGhd7 is a major negative regulator of flowering, binding to the promoter motif TGAATG(A/T)(A/T/C) and repressing transcription of the major florigen FLOWERING LOCUS T 10 (SbFT10) and floral activators EARLY HEADING DATE (SbEhd1), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (SbFKF1) and EARLY FLOWERING 3 (SbELF3). Reinforcing the direct effect of SbGhd7, SbEhd1 OE activated the promoters of three functional florigens (SbFT1, SbFT8 and SbFT10), dramatically accelerating flowering.
Our studies demonstrate that SbGhd7 is a major repressor of sorghum flowering by directly and indirectly targeting genes for flowering activation. The mechanism appears ancient. Our study extends the current model of floral transition regulation in sorghum and provides a framework for a comprehensive understanding of sorghum photoperiod response.
https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.19591
Xiong, J., Liu, Y., Wu, P. et al. (2024): Identification and virus-induced gene silencing (VIGS) analysis of methyltransferase
affecting tomato (Solanum lycopersicum) fruit ripening. Planta 259, 109 | https://doi.org/10.1007/s00425-024-04384-4
Fruit ripening is a critical stage for the formation of edible quality and seed maturation, which is finely modulated by kinds of factors, including genetic regulators, hormones, external signals, etc. Methyltransferases (MTases), important genetic regulators, play vital roles in plant development through epigenetic regulation, post-translational modification, or other mechanisms. However, the regulatory functions of numerous MTases except DNA methylation in fruit ripening remain limited so far. Here, six MTases, which act on different types of substrates, were identified to affect tomato fruit ripening. First, 35 MTase genes with relatively high expression at breaker (Br) stage of tomato fruit were screened from the tomato MTase gene database encompassing 421 genes totally. Thereafter, six MTase genes were identified as potential regulators of fruit ripening via virus-induced gene silencing (VIGS), including four genes with a positive regulatory role and two genes with a negative regulatory role, respectively. The expression of these six MTase genes exhibited diverse patterns during the fruit ripening process, and responded to various external ripening-related factors, including ethylene, 1-methylcyclopropene (1-MCP), temperature, and light exposure. These results help to further elaborate the biological mechanisms of MTase genes in tomato fruit ripening and enrich the understanding of the regulatory mechanisms of fruit ripening involving MTases, despite of DNA MTases.
https://link.springer.com/article/10.1007/s00425-024-04384-4
Ganaparthi, V.R., Wechter, P., Levi, A. et al. (2024): Mapping and validation of Fusarium wilt race 2 resistance QTL from
Citrullus amarus line USVL246-FR2. Theor Appl Genet 137, 91 | https://doi.org/10.1007/s00122-024-04595-z
Fusarium oxysporum f. sp. niveum (Fon) race 2 causes Fusarium wilt in watermelon and threatens watermelon production worldwide. Chemical management options are not effective, and no resistant edible watermelon cultivars have been released. Implementation of marker-assisted selection to develop resistant cultivars requires identifying sources of resistance and the underlying quantitative trait loci (QTL), developing molecular markers associated with the QTL, and validating marker-phenotype associations with an independent population. An intraspecific Citrullus amarus recombinant inbred line population from a cross of resistant USVL246-FR2 and susceptible USVL114 was used for mapping Fon race 2 resistance QTL. KASP markers were developed (N = 51) for the major QTL on chromosome 9 and minor QTL on chromosomes 1, 6, and 8. An interspecific F2:3 population was developed from resistance donor USVL246-FR2 (C. amarus) and a susceptible cultivar ‘Sugar Baby’ (Citrullus lanatus) to validate the utility of the markers for introgression of resistance from the wild crop relative into cultivated watermelon. Only 16 KASP markers segregated in the interspecific C. amarus/lanatus validation population. Four markers showed significant differences in the separation of genotypes based on family-mean disease severity, but together explained only 16% of the phenotypic variance. Genotypes that inherited homozygous resistant parental alleles at three KASP markers had 42% lower family-mean disease severity than homozygous susceptible genotypes. Thus, haplotype analysis was more effective at predicting the mean disease severity of families than single markers. The haplotype identified in this study will be valuable for developing Fon race 2 resistant watermelon cultivars.
https://link.springer.com/article/10.1007/s00122-024-04595-z
Nousias, O., Zheng, J., Li, T. et al. (2024): Three de novo assembled wild cacao genomes from the Upper Amazon.
Sci Data 11, 369 (2024). https://doi.org/10.1038/s41597-024-03215-1
Theobroma cacao, the chocolate tree, is indigenous to the Amazon basin, the greatest biodiversity hotspot on earth. Recent advancement in plant genomics highlights the importance of de novo sequencing of multiple reference genomes to capture the genome diversity present in different cacao populations. In this study, three high-quality chromosome-level genomes of wild cacao were constructed, de novo assembled with HiFi long reads sequencing, and scaffolded using a reference-free strategy. These genomes represent the three most important genetic clusters of cacao trees from the Upper Amazon region. The three wild cacao genomes were compared with two reference genomes of domesticated cacao. The five cacao genetic clusters were inferred to have diverged in the early and middle Pleistocene period, approximately 1.83–0.69 million years ago. The results shown here serve as an example of understanding how the Amazonian biodiversity was developed. The three wild cacao genomes provide valuable resources for studying genetic diversity and advancing genetic improvement of this species.
https://www.nature.com/articles/s41597-024-03215-1
Suchy, F.P., Karigane, D., Nakauchi, Y. et al. (2024): Genome engineering with Cas9 and AAV repair templates generates
frequent concatemeric insertions of viral vectors. Nat Biotechnol https://doi.org/10.1038/s41587-024-02171-w
CRISPR–Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells. Although these highly abundant errors were found in more than half of the edited cells, they could not be readily detected by common analytical methods. We describe strategies to detect and thoroughly characterize the concatemeric viral vector insertions, and we highlight analytical pitfalls that mask their prevalence. We then describe strategies to prevent the concatemeric inserts by cutting the vector genome after transduction. This approach is compatible with established gene editing pipelines, enabling robust genetic knockins that are safer, more reliable and more reproducible.
https://www.nature.com/articles/s41587-024-02171-w
Colombi E., Bertels F., Doulcier G., +5, and Rainey P.B. (2024): Rapid dissemination of host metabolism–manipulating genes
via integrative and conjugative elements. PNAS 121 (11) e2309263121 | https://doi.org/10.1073/pnas.2309263121
Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa. PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn6212. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn6212-encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.
https://www.pnas.org/doi/10.1073/pnas.2309263121
Genetically encoding colors and images into bioengineered microbial materials.
Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02200-8
Using synthetic biology, we engineered a cellulose-producing bacterium that can produce eumelanin and respond to light, so that it is possible to grow a microbial leather material that is colored black or contains projected black patterns.
https://www.nature.com/articles/s41587-024-02200-8
Fraiture M.-A., Gobbo A., Guillitte C., Marchesi U. et al. (2024): Pilot market surveillance of GMM contaminations in alpha-
amylase food enzyme products: A detection strategy strengthened by a newly developed qPCR method targeting a GM Bacillus licheniformis producing alpha-amylase. Food Chemistry: Molecular Sciences 8, 100186
Using high-throughput metagenomics on commercial microbial fermentation products, DNA from a new unauthorized genetically modified microorganism (GMM), namely the GM B. licheniformis strain producing alpha-amylase (GMM alpha-amylase2), was recently discovered and characterized. On this basis, a new qPCR method targeting an unnatural association of sequences specific to the GMM alpha-amylase2 strain was designed and developed in this study, allowing to strengthen the current GMM detection strategy. The performance of the newly developed qPCR method was assessed for its specificity and sensitivity to comply with the minimum performance requirements established by the European Network of GMO Laboratories for GMO analysis. Moreover, the transferability of the in house validated qPCR method was demonstrated. Finally, its applicability was confirmed by a pilot market surveillance of GMM contaminations conducted for the first time on 40 alpha-amylase food enzyme products labelled as containing alpha-amylase. This pilot market surveillance allowed also to highlight numerous contaminations with GMM alpha-amylase2, including frequent cross-contaminations with other GMM strains previously characterized. In addition, the presence of full-length AMR genes, raising health concerns, was also reported.
https://www.sciencedirect.com/science/article/pii/S2666566223000266#s0010
EFSA
CEP Panel (2024): Safety evaluation of an extension of use of the food enzyme triacylglycerol lipase from the non-genetically
modified Mucor circinelloides strain AE-LMH. EFSA Journal, 22(4), e8699. https://doi.org/10.2903/j.efsa.2024.8699
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2024.8699
CEP Panel (2024): Safety evaluation of an extension of use of the food enzyme α-glucosidase from the non-genetically modified
Aspergillus niger strain AE-TGU. EFSA Journal, 22(4), e8697. https://doi.org/10.2903/j.efsa.2024.8697
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2024.8697
CEP Panel (2024): Safety evaluation of the food enzyme AMP deaminase from non-genetically modified Aspergillus sp. strain DEA
56-111. EFSA Journal, 22(4), e8718. https://doi.org/10.2903/j.efsa.2024.8718
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2024.8718
CEP Panel (2024): Safety evaluation of the food enzyme preparation D-psicose 3-epimerase from the non-genetically modified
Microbacterium foliorum strain SYG27B. EFSA Journal, 22(4), e8702. https://doi.org/10.2903/j.efsa.2024.8702
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2024.8702
CEP Panel (2024): Safety evaluation of the food enzyme bacillolysin from the non-genetically modified Bacillus amyloliquefaciens
strain AE-NP. EFSA Journal, 22(4), e8710. https://doi.org/10.2903/j.efsa.2024.8710
https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2024.8710