Maria A. Lebedeva, Oksana L. Razhina, Veronica V. Nikanorkina, V. Taranov
The most common application of CRISPR-Cas9 genome editing system is a gene knockout via indel mutations introducing. It is obvious, because this approach has minimum critical conditions in guide RNA design: available PAM sequence and conservative 19–25 nucleotides within all alleles of a target gene. Precise nucleotide changing with base editing systems has more limitations: target nucleotide should locate in editing window of adenine- or cytidine-deaminase, besides this, all undesired adenines or cytosines in editing window will be likely changed. However, there is a more fundamental issue — it is very difficult to find a single aminoacid substitution, which changes protein features in a desirable side. One of the good examples of base editing target will be considered in this work. Nicotiana tabacumL. is a plant fromSolanaceaefamily, the same as potato, tomato and pepper. All these plants are strongly affected by potato virus Y (PVY). It is known, that PVY recruits host translation initiation factor eIF4E by the viral protein VPg in order to start synthesis its proteins. If eIF4E can’t interact with VPg, plant will be resistant. In our work, we established an aminoacid substitution in tobacco eIF4E factor, which disrupted interaction with PVY VPg in yeast two-hybrid conditions, but didn’t influence the factor functionality. Then we designed two genetic constructions with different sgRNAs for introducing this mutation in tobacco plants using cytidine-deaminase system. These constructions were used to plant transformation and development of edited tobacco plants. This work is supported by State Task No. 0431-2022-0004.
{"title":"The strong base for using base editing in plants","authors":"Maria A. Lebedeva, Oksana L. Razhina, Veronica V. Nikanorkina, V. Taranov","doi":"10.17816/ecogen567885","DOIUrl":"https://doi.org/10.17816/ecogen567885","url":null,"abstract":"The most common application of CRISPR-Cas9 genome editing system is a gene knockout via indel mutations introducing. It is obvious, because this approach has minimum critical conditions in guide RNA design: available PAM sequence and conservative 19–25 nucleotides within all alleles of a target gene. Precise nucleotide changing with base editing systems has more limitations: target nucleotide should locate in editing window of adenine- or cytidine-deaminase, besides this, all undesired adenines or cytosines in editing window will be likely changed. However, there is a more fundamental issue — it is very difficult to find a single aminoacid substitution, which changes protein features in a desirable side. One of the good examples of base editing target will be considered in this work. \u0000Nicotiana tabacumL. is a plant fromSolanaceaefamily, the same as potato, tomato and pepper. All these plants are strongly affected by potato virus Y (PVY). It is known, that PVY recruits host translation initiation factor eIF4E by the viral protein VPg in order to start synthesis its proteins. If eIF4E can’t interact with VPg, plant will be resistant. \u0000In our work, we established an aminoacid substitution in tobacco eIF4E factor, which disrupted interaction with PVY VPg in yeast two-hybrid conditions, but didn’t influence the factor functionality. Then we designed two genetic constructions with different sgRNAs for introducing this mutation in tobacco plants using cytidine-deaminase system. These constructions were used to plant transformation and development of edited tobacco plants. \u0000This work is supported by State Task No. 0431-2022-0004.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"11 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603184","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sonya Sokornova, Maryna N. Mandrik-Litvinkovich, T. Matveeva
Root endophytic fungi (EF) spend at least parts of their life cycle inside plant tissues without apparent harm to the host. There is a hypothesis that the endophytic lifestyle is a common strategy for most fungi and they have endophytic ancestors [1]. By receiving habitat and nutrients EF can increase the solubility of nutrients in the plant rhizosphere, stimulate plant growth, and activate the plant’s systemic resistance to stress. One of the alternatives to the use of pesticides is the use of resistant transgenic plants, but the potential effects of crop modifications on their associated microorganisms are poorly studied. The EF communities of transgenic lines of cotton, sugar cane, and maize containing the expressed Cry1 protein from Bacillus thuringiensis were compared with communities of non-transgenic plants. There were no significant differences in the composition of the EF community [2, 3]. The introduction of phosphinothricin-N-acetyltransferase and imazapyr herbicide resistance genes for corn and sugar cane also did not affect on EF communities but did affect the bacterial community [3, 4]. The similar effect was observed for transgenic maple plants [5]. The stage of plant development had a more significant effect on EF community than the fact of transformation itself [1]. We believe that the fungal community is more conservative and the introduction of herbicide resistance or toxin synthesis genes into the plant genome has a significantly lesser effect on EF community than on the bacterial one.
{"title":"Characteristics of root endophytic fungi communities associated with genetically modified plants","authors":"Sonya Sokornova, Maryna N. Mandrik-Litvinkovich, T. Matveeva","doi":"10.17816/ecogen568501","DOIUrl":"https://doi.org/10.17816/ecogen568501","url":null,"abstract":"Root endophytic fungi (EF) spend at least parts of their life cycle inside plant tissues without apparent harm to the host. There is a hypothesis that the endophytic lifestyle is a common strategy for most fungi and they have endophytic ancestors [1]. By receiving habitat and nutrients EF can increase the solubility of nutrients in the plant rhizosphere, stimulate plant growth, and activate the plant’s systemic resistance to stress. One of the alternatives to the use of pesticides is the use of resistant transgenic plants, but the potential effects of crop modifications on their associated microorganisms are poorly studied. \u0000The EF communities of transgenic lines of cotton, sugar cane, and maize containing the expressed Cry1 protein from Bacillus thuringiensis were compared with communities of non-transgenic plants. There were no significant differences in the composition of the EF community [2, 3]. The introduction of phosphinothricin-N-acetyltransferase and imazapyr herbicide resistance genes for corn and sugar cane also did not affect on EF communities but did affect the bacterial community [3, 4]. The similar effect was observed for transgenic maple plants [5]. The stage of plant development had a more significant effect on EF community than the fact of transformation itself [1]. \u0000We believe that the fungal community is more conservative and the introduction of herbicide resistance or toxin synthesis genes into the plant genome has a significantly lesser effect on EF community than on the bacterial one.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"3 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anastasia S. Makeeva, A. Sidorin, Valeriia V. Ishtuganova, M. Padkina, A. M. Rumyantsev
Komagataella phaffii(Pichia pastoris) is known to be an excellent producer of recombinant proteins for industrial and research purposes. Protein synthesis improvement in this yeast includes selection of optimal cultivation parameters [1, 2]. Therefore, much attention is paid to the influence of media components on physiology of this yeast [3–5]. One of the essential media components is biotin. In yeast cells it plays a crucial role as a cofactor of enzymes, providing carboxylation reactions in lipo-, gluconeogenesis, and nitrogen metabolism.K. phaffiiis biotin auxotrophic organism unable to synthesize this vitaminde novo. Thus, it necessarily requires adding of biotin in the media. In this study, we analyzed the effect of biotin starvation on gene expression inK. phaffiicells during its growth on methanol- and glycerin-containing media. These carbon sources are the most commonly used in standard protocols for recombinant protein biosynthesis inK. phaffii. It was shown, that biotin starvation cell response significantly depends on carbon source. In glycerol-containing media biotin deficiency enhanced transcription of genes involved in biotin and thiamine metabolism, glyoxylate cycle, synthesis of acetyl-CoA in cytoplasm and its carnitine-mediated transport into mitochondria. Genes involved in biosynthesis of lipids and glucose were repressed in media with glycerol. In methanol-containing media the biotin deficiency effect was more pronounced and led to repression of numerous genes involved in protein and amino acids synthesis and activation of cell response to oxidative stress. The obtained results are thought to be important for optimizing the culture conditions in theK. phaffiiexpression systems.
{"title":"Effect of biotin starvation on gene expression in industrially significant yeast Komagataella phaffii","authors":"Anastasia S. Makeeva, A. Sidorin, Valeriia V. Ishtuganova, M. Padkina, A. M. Rumyantsev","doi":"10.17816/ecogen568379","DOIUrl":"https://doi.org/10.17816/ecogen568379","url":null,"abstract":"Komagataella phaffii(Pichia pastoris) is known to be an excellent producer of recombinant proteins for industrial and research purposes. Protein synthesis improvement in this yeast includes selection of optimal cultivation parameters [1, 2]. Therefore, much attention is paid to the influence of media components on physiology of this yeast [3–5]. \u0000One of the essential media components is biotin. In yeast cells it plays a crucial role as a cofactor of enzymes, providing carboxylation reactions in lipo-, gluconeogenesis, and nitrogen metabolism.K. phaffiiis biotin auxotrophic organism unable to synthesize this vitaminde novo. Thus, it necessarily requires adding of biotin in the media. \u0000In this study, we analyzed the effect of biotin starvation on gene expression inK. phaffiicells during its growth on methanol- and glycerin-containing media. These carbon sources are the most commonly used in standard protocols for recombinant protein biosynthesis inK. phaffii. \u0000It was shown, that biotin starvation cell response significantly depends on carbon source. In glycerol-containing media biotin deficiency enhanced transcription of genes involved in biotin and thiamine metabolism, glyoxylate cycle, synthesis of acetyl-CoA in cytoplasm and its carnitine-mediated transport into mitochondria. Genes involved in biosynthesis of lipids and glucose were repressed in media with glycerol. In methanol-containing media the biotin deficiency effect was more pronounced and led to repression of numerous genes involved in protein and amino acids synthesis and activation of cell response to oxidative stress. \u0000The obtained results are thought to be important for optimizing the culture conditions in theK. phaffiiexpression systems.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"25 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The burgeoning field of nanotechnology has witnessed a surge in the utilization of biological entities, especially plant extracts, for the green synthesis of nanoparticles. In this innovative study, we have ventured into the realm of genetic engineering to optimize the synthesis of silver nanoparticles (AgNPs) usingDaturametel, a plant traditionally known for its rich phytoconstituents [1, 2]. Our initial experiments with non-modifiedDatura metelfruit extracts as reducing agents yielded AgNPs with an average size of 40–50 nm, confirmed spectrophotometrically with a peak at 460 nm. Recognizing the potential to enhance this process, we genetically modifiedDatura metelplants to amplify their phytoconstituent content by approximately 30%. This was achieved by overexpressing genes associated with the production of specific phytochemicals, such as polyphenols and amides. Subsequent synthesis processes using the GMDatura metelextracts resulted in a 25% increase in nanoparticle yield. Furthermore, the average size of the nanoparticles synthesized from GM extracts ranged between 20–30 nm, indicating a more uniform and refined synthesis process. Advanced analytical techniques, including X-ray diffraction, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and Energy-dispersive X-ray spectroscopy (EDX), were employed to validate these findings. Notably, the EDX analysis of nanoparticles synthesized from GM extracts showcased a silver peak contributing to 32–35% of the weight, a slight increase from the non-modified counterparts. Beyond the quantitative enhancements, the GM approach also influenced the qualitative properties of the AgNPs. Preliminary tests indicate that the nanoparticles derived from GM extracts exhibit enhanced antimicrobial and antioxidant properties, making them potential candidates for various biomedical applications. In conclusion, this study underscores the immense potential of integrating genetic engineering with nanotechnology. By genetically enhancingDatura metel, we have not only optimized the synthesis process of AgNPs but also broadened the horizons for their potential applications. However, as we advance in this direction, it is imperative to tread with caution, ensuring the ethical and safe use of GM organisms in research and applications.
{"title":"Genetic enhancement of Datura metel for optimized silver nanoparticle synthesis","authors":"S. Meenakshi, A. Delta, P. Kaushik","doi":"10.17816/ecogen568587","DOIUrl":"https://doi.org/10.17816/ecogen568587","url":null,"abstract":"The burgeoning field of nanotechnology has witnessed a surge in the utilization of biological entities, especially plant extracts, for the green synthesis of nanoparticles. In this innovative study, we have ventured into the realm of genetic engineering to optimize the synthesis of silver nanoparticles (AgNPs) usingDaturametel, a plant traditionally known for its rich phytoconstituents [1, 2]. \u0000Our initial experiments with non-modifiedDatura metelfruit extracts as reducing agents yielded AgNPs with an average size of 40–50 nm, confirmed spectrophotometrically with a peak at 460 nm. Recognizing the potential to enhance this process, we genetically modifiedDatura metelplants to amplify their phytoconstituent content by approximately 30%. This was achieved by overexpressing genes associated with the production of specific phytochemicals, such as polyphenols and amides. \u0000Subsequent synthesis processes using the GMDatura metelextracts resulted in a 25% increase in nanoparticle yield. Furthermore, the average size of the nanoparticles synthesized from GM extracts ranged between 20–30 nm, indicating a more uniform and refined synthesis process. Advanced analytical techniques, including X-ray diffraction, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and Energy-dispersive X-ray spectroscopy (EDX), were employed to validate these findings. Notably, the EDX analysis of nanoparticles synthesized from GM extracts showcased a silver peak contributing to 32–35% of the weight, a slight increase from the non-modified counterparts. \u0000Beyond the quantitative enhancements, the GM approach also influenced the qualitative properties of the AgNPs. Preliminary tests indicate that the nanoparticles derived from GM extracts exhibit enhanced antimicrobial and antioxidant properties, making them potential candidates for various biomedical applications. \u0000In conclusion, this study underscores the immense potential of integrating genetic engineering with nanotechnology. By genetically enhancingDatura metel, we have not only optimized the synthesis process of AgNPs but also broadened the horizons for their potential applications. However, as we advance in this direction, it is imperative to tread with caution, ensuring the ethical and safe use of GM organisms in research and applications.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"18 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138603928","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Modern bioengineering technologies make it possible to create fruit and berry crops with signs that are unattainable by traditional breeding methods, while significantly reducing the time of breeding work. A review of more than 90 scientific publications in the period from 1989 to 2020 with research work on the creation of transgenic fruit and berry crops using various bioengineering technologies, which showed a wide variety of methods for modifying and identifying the resulting plants. For example, the modification of the genome of an apple tree using genes that accelerate flowering, the so-called “Fast breeding” technology, allows you to speed up breeding work to create valuable varieties. The developed new varieties of transgenic apple trees are resistant to the fungi, bacteria, apple moth, and also reduced ability to browning fruits, with an increased content of sucrose, etc. Modern methods of genetic engineering make it possible to significantly accelerate the processes of creating highly productive varieties of fruit crops with increased or complete resistance to viruses. Transgenic papaya, expressing the gene for the envelope protein of the virus, made it possible to save plantations in Hawaii. The plum varieties created with the help of bioengineering were resistant to the plum pox virus (PPV), which poses a greater danger to perennial fruit crops. In Russia, clonal rootstocks of apple and pear trees resistant to herbicides, strawberries and pears with improved fruit taste, rootstocks and varieties of plums and cherries resistant to the Plum Pox Virus have been created and field tested, a technology for obtaining cisgenic plants has been developed and applied using the example of apple and tomato trees that do not contain viral and bacterial sequences. Methods of genomic editing and accelerated selection of fruit crops are being developed.
{"title":"Bioengineering of horticultural crops in Russia and in the world","authors":"Sergey V. Dolgov","doi":"10.17816/ecogen568614","DOIUrl":"https://doi.org/10.17816/ecogen568614","url":null,"abstract":"Modern bioengineering technologies make it possible to create fruit and berry crops with signs that are unattainable by traditional breeding methods, while significantly reducing the time of breeding work. A review of more than 90 scientific publications in the period from 1989 to 2020 with research work on the creation of transgenic fruit and berry crops using various bioengineering technologies, which showed a wide variety of methods for modifying and identifying the resulting plants. \u0000For example, the modification of the genome of an apple tree using genes that accelerate flowering, the so-called “Fast breeding” technology, allows you to speed up breeding work to create valuable varieties. The developed new varieties of transgenic apple trees are resistant to the fungi, bacteria, apple moth, and also reduced ability to browning fruits, with an increased content of sucrose, etc. \u0000Modern methods of genetic engineering make it possible to significantly accelerate the processes of creating highly productive varieties of fruit crops with increased or complete resistance to viruses. Transgenic papaya, expressing the gene for the envelope protein of the virus, made it possible to save plantations in Hawaii. The plum varieties created with the help of bioengineering were resistant to the plum pox virus (PPV), which poses a greater danger to perennial fruit crops. \u0000In Russia, clonal rootstocks of apple and pear trees resistant to herbicides, strawberries and pears with improved fruit taste, rootstocks and varieties of plums and cherries resistant to the Plum Pox Virus have been created and field tested, a technology for obtaining cisgenic plants has been developed and applied using the example of apple and tomato trees that do not contain viral and bacterial sequences. Methods of genomic editing and accelerated selection of fruit crops are being developed.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"78 24","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138604728","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Horizontal gene transfer from agrobacteria to plants turned out to be a much more widespread phenomenon than previously thought. In the first it was established in 2019 due to the application of bioinformatic methods and databases, then about 30 species of naturally transgenic plants were discovered [1]. The deposition of new nucleotide sequences of various plant species makes it possible to periodically update the list of naturally GMOs. Analysis of genomic and transcriptomic databases in 2023 revealed more than 50 new naturally transgenic plants and, thus, more than 100 species of nGMOs are currently known. And the share of naturally transgenic plants in relation to deposited species of terrestrial dicotyledonous plants is about 7%. Interestingly, this indicator retains its value regardless of the change in the number of organisms in the studied databases [2]. Among the discovered new nGMOs there are species that have been cultivated by humans since ancient times and are important agricultural crops. Fruit crops include the following species of nGMOs: carambola (Averrhoa carambola L.), persimmon (Diospyros kaki Thunb.), wasabi (Eutrema japonicum (Miq.) Koidz.), raspberry (Rubus idaeus L.), Luffa acutangula (L.) Roxb. There are also many medicinal, ornamental and oilseed species among the naturally transgenic plants. Further study of these species would make it possible to establish what role horizontal gene transfer played in the appearance of traits in plants that were selected by humans. The obtained data can be further used to study the molecular evolution and the role of transgenes in naturally transgenic plants.
从农杆菌到植物的水平基因转移被证明是一个比以前认为的更广泛的现象。首先是在2019年通过应用生物信息学方法和数据库建立，随后发现了约30种天然转基因植物[1]。各种植物新核苷酸序列的沉积使得定期更新天然转基因生物列表成为可能。在2023年的基因组和转录组数据库分析中发现了50多种新的天然转基因植物，因此目前已知的nGMOs超过100种。天然转基因植物占陆生双子叶植物沉积种的7%左右。有趣的是，无论研究数据库中生物数量的变化如何，该指标都保持其价值[2]。在新发现的转基因生物中，有一些自古以来就被人类种植的物种，是重要的农作物。水果作物包括以下几种转基因生物:杨桃(Averrhoa carambola L.)、柿子(Diospyros kaki Thunb.)、山葵(Eutrema japonicum (Miq.))。树莓(Rubus idaeus L.)、丝瓜(Luffa acutangula)Roxb。在天然转基因植物中，还有许多药用、观赏和油籽类植物。对这些物种的进一步研究将有可能确定水平基因转移在人类选择的植物性状的出现中所起的作用。所获得的数据可以进一步用于研究基因在自然转基因植物中的分子进化和作用。
{"title":"New naturally transgenic crop","authors":"Anton D. Shaposhnikov, T. Matveeva","doi":"10.17816/ecogen568608","DOIUrl":"https://doi.org/10.17816/ecogen568608","url":null,"abstract":"Horizontal gene transfer from agrobacteria to plants turned out to be a much more widespread phenomenon than previously thought. In the first it was established in 2019 due to the application of bioinformatic methods and databases, then about 30 species of naturally transgenic plants were discovered [1]. The deposition of new nucleotide sequences of various plant species makes it possible to periodically update the list of naturally GMOs. Analysis of genomic and transcriptomic databases in 2023 revealed more than 50 new naturally transgenic plants and, thus, more than 100 species of nGMOs are currently known. And the share of naturally transgenic plants in relation to deposited species of terrestrial dicotyledonous plants is about 7%. Interestingly, this indicator retains its value regardless of the change in the number of organisms in the studied databases [2]. \u0000Among the discovered new nGMOs there are species that have been cultivated by humans since ancient times and are important agricultural crops. Fruit crops include the following species of nGMOs: carambola (Averrhoa carambola L.), persimmon (Diospyros kaki Thunb.), wasabi (Eutrema japonicum (Miq.) Koidz.), raspberry (Rubus idaeus L.), Luffa acutangula (L.) Roxb. There are also many medicinal, ornamental and oilseed species among the naturally transgenic plants. Further study of these species would make it possible to establish what role horizontal gene transfer played in the appearance of traits in plants that were selected by humans. \u0000The obtained data can be further used to study the molecular evolution and the role of transgenes in naturally transgenic plants.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"49 6","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138602218","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anastasiya M. Artemiuk, V. Tvorogova, Lyudmila A. Lutova
Somatic embryogenesis is the formation of embryos from plant’s somatic cells. It is widely used in biotechnology for reproduction of plants, studying of regeneration process and it also represents a convenient way to obtain transgenic plants. Currently, a solid medium is usually used for the formation of transgenic somatic embryos, which has a number of disadvantages. We are developing a system for cultivating explants in a liquid medium for the transformation and formation of somatic embryos forMedicago truncatula. Unlike a solid medium, it should allow using petioles as explants, simplify the renewal of the medium, replace disposable cultivation containers with reusable ones, and also reduce the time required for the formation of somatic embryos. Currently, the optimal concentration of hygromycin as a selective agent in such a system was found. Interestingly, it appeared to be lower than the selective hygromycin concentration in a solid medium. The addition of cefotaxime to the medium reduces the number of somatic embryos formed, but does not completely suppress their formation. Thus, cefotaxime can be used to eliminate agrobacteria during transformation using this cultivation system. Embryos withGUSoverexpression transformed with this method were successfully obtained.
{"title":"Development of a system for the formation of transgenic somatic embryos in the liquid medium in Medicago truncatula","authors":"Anastasiya M. Artemiuk, V. Tvorogova, Lyudmila A. Lutova","doi":"10.17816/ecogen568297","DOIUrl":"https://doi.org/10.17816/ecogen568297","url":null,"abstract":"Somatic embryogenesis is the formation of embryos from plant’s somatic cells. It is widely used in biotechnology for reproduction of plants, studying of regeneration process and it also represents a convenient way to obtain transgenic plants. Currently, a solid medium is usually used for the formation of transgenic somatic embryos, which has a number of disadvantages. \u0000We are developing a system for cultivating explants in a liquid medium for the transformation and formation of somatic embryos forMedicago truncatula. Unlike a solid medium, it should allow using petioles as explants, simplify the renewal of the medium, replace disposable cultivation containers with reusable ones, and also reduce the time required for the formation of somatic embryos. \u0000Currently, the optimal concentration of hygromycin as a selective agent in such a system was found. Interestingly, it appeared to be lower than the selective hygromycin concentration in a solid medium. \u0000The addition of cefotaxime to the medium reduces the number of somatic embryos formed, but does not completely suppress their formation. Thus, cefotaxime can be used to eliminate agrobacteria during transformation using this cultivation system. \u0000Embryos withGUSoverexpression transformed with this method were successfully obtained.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"82 10","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138604708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
I. V. Yakovleva, Sofya E. Gaidukova, A. M. Kamionskaya
The technologies of genome editing and synthetic biology are becoming more and more accessible today and, in combination with the application of artificial intelligence in biotechnology, especially powerful. A feature of today’s stage is the rapidly changing landscape of engineering biological systems, which requires revision and updating of the biosafety framework. The proposed new oversight measures are as follows: а) screening for DNA synthesis orders and sequences of concern; b) environmental metagenome sequencing to search for synthetic organisms [1]. At the same time, DNA ‘printers’ are appeared on the market today, that blurs the boundaries of access to synthetic DNA. It is significant that no government currently requires screening or regulates it, and this system works on a benevolent basis. Additionally environmental surveillance requires for a long time to define base line. With the new scale of human activity, new social risks also arise: new forms of discrimination and inequality, confidentiality of personal data in biotechnology projects, multiplication of biotechnology and artificial intelligence risks. Thus, the idea of “responsible researches and innovation” (RRI) [2], and trend to address safety early at the concept stage — “Safe by design” have come into the focus. A number of RRI principles can be formulated at the proof concept stage for a genome- edited project: benefits for most citizens; transparency, the public comment cycle prior to the start of the experiments; responsibility, precautions, liability; justice, redress; well-being, social good.
基因组编辑和合成生物学技术在今天变得越来越容易获得，并且与人工智能在生物技术中的应用相结合，特别强大。当今阶段的一个特点是工程生物系统的迅速变化，这需要修订和更新生物安全框架。拟议的新监督措施如下:筛选DNA合成顺序和关注序列;B)环境宏基因组测序以寻找合成生物[1]。与此同时，如今市场上出现了DNA“打印机”，这模糊了获取合成DNA的界限。重要的是，目前没有政府要求对其进行筛选或监管，而且这一体系是在善意的基础上运作的。此外，环境监测需要很长时间才能确定基线。随着人类活动的新规模，新的社会风险也出现了:新形式的歧视和不平等，生物技术项目中个人数据的保密性，生物技术和人工智能风险的倍增。因此，“负责任的研究和创新”(responsible research and innovation, RRI)的理念[2]，以及在概念阶段早期就解决安全问题的趋势——“设计安全”(Safe by design)成为人们关注的焦点。在基因组编辑计划的概念验证阶段，可以制定一些RRI原则:对大多数公民有利;透明度，实验开始前的公众评论周期;责任、注意事项、责任;正义,纠正;幸福，社会福利。
{"title":"Social and ethical component of genetic technologies","authors":"I. V. Yakovleva, Sofya E. Gaidukova, A. M. Kamionskaya","doi":"10.17816/ecogen567811","DOIUrl":"https://doi.org/10.17816/ecogen567811","url":null,"abstract":"The technologies of genome editing and synthetic biology are becoming more and more accessible today and, in combination with the application of artificial intelligence in biotechnology, especially powerful. A feature of today’s stage is the rapidly changing landscape of engineering biological systems, which requires revision and updating of the biosafety framework. The proposed new oversight measures are as follows: а) screening for DNA synthesis orders and sequences of concern; b) environmental metagenome sequencing to search for synthetic organisms [1]. At the same time, DNA ‘printers’ are appeared on the market today, that blurs the boundaries of access to synthetic DNA. It is significant that no government currently requires screening or regulates it, and this system works on a benevolent basis. Additionally environmental surveillance requires for a long time to define base line. With the new scale of human activity, new social risks also arise: new forms of discrimination and inequality, confidentiality of personal data in biotechnology projects, multiplication of biotechnology and artificial intelligence risks. \u0000Thus, the idea of “responsible researches and innovation” (RRI) [2], and trend to address safety early at the concept stage — “Safe by design” have come into the focus. A number of RRI principles can be formulated at the proof concept stage for a genome- edited project: benefits for most citizens; transparency, the public comment cycle prior to the start of the experiments; responsibility, precautions, liability; justice, redress; well-being, social good.","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"17 13","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138601773","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function [1, 2]. A common construct for efficient genome editing and selection of hairy roots is comprised of three components, i.e., a cassette carrying the gene encoding the Cas nuclease, a cassette expressing the guide RNA (gRNA), and a cassette encoding a screenable or selectable marker [2]. After design and construction, the resulting vector is used to transform plant using appropriateRhizobium rhizogenesstrain. Over 26 plant species have been used in experiments combining genome editing and hairy root transformation to date [2]. Possible applications of CRISPR/Cas9 genome editing using hairy root transformation include different directions like test the efficiency of the CRISPR/Cas9 genome editing; obtaining whole genome-edited plants regenerated from individual edited hairy roots; investigation of root development or root function, root nodule symbiosis, resistance to biotic or abiotic stresses, or metabolic engineering [2]. The basic principles of plant CRISPR/Cas genome editing like the different components of CRISPR/Cas vectors, the types of Cas nuclease, design principles of gRNAs, as well as the possible applications of CRISPR/Cas genome editing in hairy roots will discuss. The application of this method for multigene editing strategy will also be demonstrated onDEEPER ROOTING1genes of cucumber. The study was supported by the Ministry of Science and Higher Education of the Russian Federation (Grant No. 075-15-2021-1056).
{"title":"Study of functional features of plant root systems using CRISPR/Cas-mediated genome editing","authors":"A. S. Kiryushkin, E. Ilina, K. Demchenko","doi":"10.17816/ecogen568351","DOIUrl":"https://doi.org/10.17816/ecogen568351","url":null,"abstract":"CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function [1, 2]. \u0000A common construct for efficient genome editing and selection of hairy roots is comprised of three components, i.e., a cassette carrying the gene encoding the Cas nuclease, a cassette expressing the guide RNA (gRNA), and a cassette encoding a screenable or selectable marker [2]. After design and construction, the resulting vector is used to transform plant using appropriateRhizobium rhizogenesstrain. \u0000Over 26 plant species have been used in experiments combining genome editing and hairy root transformation to date [2]. Possible applications of CRISPR/Cas9 genome editing using hairy root transformation include different directions like test the efficiency of the CRISPR/Cas9 genome editing; obtaining whole genome-edited plants regenerated from individual edited hairy roots; investigation of root development or root function, root nodule symbiosis, resistance to biotic or abiotic stresses, or metabolic engineering [2]. \u0000The basic principles of plant CRISPR/Cas genome editing like the different components of CRISPR/Cas vectors, the types of Cas nuclease, design principles of gRNAs, as well as the possible applications of CRISPR/Cas genome editing in hairy roots will discuss. The application of this method for multigene editing strategy will also be demonstrated onDEEPER ROOTING1genes of cucumber. \u0000The study was supported by the Ministry of Science and Higher Education of the Russian Federation (Grant No. 075-15-2021-1056).","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"24 8","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138601937","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A. V. Chirinskaite, Andrew A. Zelinsky, J. Sopova, Elena I. Leonova
CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease fromLachnospiraceaebacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNAin vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice. In conclusion, new PAM TTAA greatly increases specificity of DNA cleavage allowing to use this system as an instrument for rapid detection if microdeletions in mice. This work was supported by a Saint Petersburg State University grant for the development of scientific research (ID 94030690) and the Genome Research Centre development program “Kurchatov Genome Centre–PNPI” (agreement No. 075-15-2019-1663)
{"title":"Development of the Cas12a-based microdeletion and microinsertion detection system","authors":"A. V. Chirinskaite, Andrew A. Zelinsky, J. Sopova, Elena I. Leonova","doi":"10.17816/ecogen568454","DOIUrl":"https://doi.org/10.17816/ecogen568454","url":null,"abstract":"CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease fromLachnospiraceaebacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNAin vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice. \u0000In conclusion, new PAM TTAA greatly increases specificity of DNA cleavage allowing to use this system as an instrument for rapid detection if microdeletions in mice. \u0000This work was supported by a Saint Petersburg State University grant for the development of scientific research (ID 94030690) and the Genome Research Centre development program “Kurchatov Genome Centre–PNPI” (agreement No. 075-15-2019-1663)","PeriodicalId":11431,"journal":{"name":"Ecological genetics","volume":"14 13","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138602278","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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