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The relationship of embryonal carcinoma (EC) cells, the stem cells of germ cell‐ or embryo‐derived teratocarcinoma tumors, to early embryonic cells came under intense scrutiny in the early 1970s when mouse chimeras were produced between EC cells and embryos. These chimeras raised tantalizing possibilities and high hopes for different areas of research. The normalization of EC cells by the embryo lent validity to their use as in vitro models for embryogenesis and indicated that they might reveal information about the relationship between malignancy and differentiation. Chimeras also showed the way for the potential introduction of genes, selected in EC cells in vitro, into the germ line of mice. Although EC cells provided material for the elucidation of early embryonic events and stimulated many studies of early molecular differentiation, after years of intense scrutiny, they fell short as the means of genetic manipulation of the germ line, although arguably they pointed the way to the development of embryonic stem (ES) cells that eventually fulfilled this goal. [ABSTRACT FROM AUTHOR]

Sterilization and castration have been synonyms for thousands of years. Making an animal sterile meant to render them incapable of producing offspring. Castration or the physical removal of the testes was discovered to be the most simple but reliable method for managing reproduction and sexual behavior in the male. Today, there continues to be global utilization of castration in domestic animals. More than six hundred million pigs are castrated every year, and surgical removal of testes in dogs and cats is a routine practice in veterinary medicine. However, modern biological research has extended the meaning of sterilization to include methods that spare testis removal and involve a variety of options, from chemical castration and immunocastration to various methods of vasectomy. This review begins with the history of sterilization, showing a direct link between its practice in man and animals. Then, it traces the evolution of concepts for inducing sterility, where research has overlapped with basic studies of reproductive hormones and the discovery of testicular toxicants, some of which serve as sterilizing agents in rodent pests. Finally, the most recent efforts to use the immune system and gene editing to block hormonal stimulation of testis function are discussed. As we respond to the crisis of animal overpopulation and strive for better animal welfare, these novel methods provide optimism for replacing surgical castration in some species. [ABSTRACT FROM AUTHOR]

Engineering of genetic networks with artificial signaling pathways (ASPs) can reprogram cellular responses and phenotypes under different circumstances for a variety of diagnostic and therapeutic purposes. However, construction of ASPs between originally independent endogenous genes in mammalian cells is highly challenging. Here we report an amplifiable RNA circuit that can theoretically build regulatory connections between any endogenous genes in mammalian cells. We harness the system of catalytic hairpin assembly with combination of controllable CRISPR‐Cas9 function to transduce the signals from distinct messenger RNA expression of trigger genes into manipulation of target genes. Through introduction of these RNA‐based genetic circuits, mammalian cells are endowed with autonomous capabilities to sense the changes of RNA expression either induced by ligand stimuli or from various cell types and control the cellular responses and fates via apoptosis‐related ASPs. Our design provides a generalized platform for construction of ASPs inside the genetic networks of mammalian cells based on differentiated RNA expression. [ABSTRACT FROM AUTHOR]

Sterilization and castration have been synonyms for thousands of years. Making an animal sterile meant to render them incapable of producing offspring. Castration or the physical removal of the testes was discovered to be the most simple but reliable method for managing reproduction and sexual behavior in the male. Today, there continues to be global utilization of castration in domestic animals. More than six hundred million pigs are castrated every year, and surgical removal of testes in dogs and cats is a routine practice in veterinary medicine. However, modern biological research has extended the meaning of sterilization to include methods that spare testis removal and involve a variety of options, from chemical castration and immunocastration to various methods of vasectomy. This review begins with the history of sterilization, showing a direct link between its practice in man and animals. Then, it traces the evolution of concepts for inducing sterility, where research has overlapped with basic studies of reproductive hormones and the discovery of testicular toxicants, some of which serve as sterilizing agents in rodent pests. Finally, the most recent efforts to use the immune system and gene editing to block hormonal stimulation of testis function are discussed. As we respond to the crisis of animal overpopulation and strive for better animal welfare, these novel methods provide optimism for replacing surgical castration in some species.

Abstract Background Filamentous fungi possess a rich CAZymes system, which is widely studied and applied in the bio-conversion of plant biomass to alcohol chemicals. Carbon source acquisition is the fundamental driver for CAZymes-producing sustainability and secondary metabolism, therefore, a deeper insight into the regulatory network of sugar transport in filamentous fungi has become urgent. Results This study reports an important linkage of sulfur assimilation to lignocellulose response of filamentous fungus. Inorganic sulfur addition facilitated biodegradation of rice straw by Trichoderma guizhouense NJAU4742. Cysteine and glutathione were revealed as major intracellular metabolites responsive to sulfur addition by metabolomics, cysteine content was increased in this process and glutathione increased correspondingly. Two membrane sugar transporter genes, Tgmst1 and Tgmst2, were identified as the critical response genes significantly up-regulated when intracellular cysteine increased. Tgmst1 and Tgmst2 were both positively regulated by the glucose regulation-related protein (GRP), up-regulation of both Tgmst1 and Tggrp can cause a significant increase in intracellular glucose. The transcriptional regulatory function of GRP mainly relied on GSH-induced glutathionylation, and the transcription activating efficiency was positively related to the glutathionylation level, furthermore, DTT-induced deglutathionylation resulted in the down-regulation of downstream genes. Conclusions Inorganic sulfur addition induces a rise in intracellular Cys content, and the conversion of cysteine to glutathione caused the increase of glutathionylation level of GRP, which in turn up-regulated Tgmst1 and Tgmst2. Subsequently, the sugar transport efficiency of single cells was improved, which facilitated the maintenance of vigorous CAZymes metabolism and the straw-to-biomass conversion.

Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized (Cno) Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (Cno CAS9 or "CnoCAS9") reliably enabled genome editing in the widely used KN99α C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.

Rice is an important dietary source of the toxic mineral cadmium (Cd) for populations in which rice is the main staple food. When grown in agricultural soils that are contaminated with Cd, rice often accumulates excessive Cd into the grains, which is a serious threat to agricultural sustainability and human health. To limit Cd accumulation in rice grains, studies on the genetic basis of Cd accumulation in rice have been carried out extensively, and some low-Cd rice varieties have also been developed in recent years. However, the challenges in low-Cd rice breeding still exist because the outcomes of the current genetic improvements for low-Cd rice cannot fully meet the requirements for the development of Cd-safe rice at present. In this review, we outline the progress in understanding the physiological mechanisms and the genetic nature of Cd accumulation in rice and summarize the strategies and outcomes of low-Cd rice breeding over the past decade. By graphing the physiological mechanism of Cd transport in the rice plant, three key steps and some underlying genes are summarized and discussed. Also, two genetic features of the natural variation in rice grain-Cd accumulation, the phenotypic plasticity and subspecies divergence, and the potential genetic explanations for these features are also discussed. Finally, we summarize and discuss current progress and the potential issues in low-Cd rice breeding using different breeding strategies. We hope to propose strategies for future success in the breeding of low-Cd rice varieties over the next decade.

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24–36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes' integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum. [ABSTRACT FROM AUTHOR]

Abstract Background The marine diatom Phaeodactylum tricornutum is a commercially viable species due to its bioactive substances and lipid productivity. Increasing attention has been paid to the isolation or genetic modification of species or strains with a rapid growth rate and large quantities of lipids. Furthermore, contamination of microzooplankton has been one of the major constraints in P. tricornutum large-scale cultivation, which adversely affects growth and greatly impedes the course of biomass production industrialization. Results Here, based on our previous transcriptomics of P. tricornutum, we found a novel gene (ID: 7202015, hereafter called Pt2015) which affects morphotype of P. tricornutum. Pt2015 protein is located in the plastid, which is highly homologous to part of the sequences of exosome component. The morphotype of the Pt2015 knockout strain (termed 2015KO) using CRISPR/Cas9 method is fusiform, but the Pt2015 overexpression strain (termed oeT) demonstrates a majority triradiate morphotype (approximately 95%) which is stable and has been cultured for more than 200 generations. In addition, the oeT strain demonstrated a similar growth rate to the WT and simultaneously accumulated larger lipids droplets that increased by approximately 30% compared to that of the WT. More importantly, the grazing rate of the amoebae cultured in the oeT strain significantly decreased in comparison with that cultured in WT, suggesting that the oeT can effectively avoid being eaten by microzooplankton. Conclusions Therefore, the oeT strain not only improves our understanding of morphotype conversion in diatoms but also demonstrates potential applications for large-scale cultivation of P. tricornutum.

Gene editing techniques, which help in modification of any DNA sequence at ease, have revolutionized the world of Genetic engineering. Although there are other gene-editing techniques, CRISPR has emerged as the chief and most preferred tool due to its simplicity and capacity to execute effective gene editing in a wide range of organisms. Although Cas9 has widely been employed for genetic modification over the years, Cas12 systems have lately emerged as a viable option. This review primarily focuses on assessing Cas12-mediated mutagenesis and elucidating the editing efficacy of both Cpf1 (Cas12a) and C2c1 (Cas12b) systems in microbes, plants, and other species. Also, we reviewed several genetic alterations that have been performed with these Cas12 systems to improve editing efficiency. Furthermore, the experimental benefits and applications of Cas12 systems are highlighted in this study. [ABSTRACT FROM AUTHOR]

• Tumor-derived EVs serve as delivery for chemotherapeutic drugs and dual genes manipulation tools. • EVs-based gene editing tool delivery strategies are efficient both for glioblastoma. • Dual genes manipulation enhanced chemotherapy potentiates antitumor immunity. Glioblastoma is one of the most malignant tumors in the central nervous system, which the conventional therapeutic modalities fail to cure the disease but improve the treatment outcome slightly. In the past decades, immune checkpoint blockade (ICB) and cytokine-regulated immunotherapy have ushered in a new era for glioblastoma treatment. However, the immunosuppressive tumor microenvironment hinders and opposites the therapeutic outcomes. Therefore, the increased importance and necessary for better modality in urgent. Moreover, many novel methods and strategies are searching for more effectively therapeutic outcomes. In this study, the glioblastoma cells derived extracellular vesicles (EVs) were adopted as a delivery platform for the chemotherapy agent (doxorubicin, Dox), gene editing platform of CD47 downregulation and IL-9 overexpression (EVs@Dox/sgCD47/IL-9), due to its excellent blood brain barrier (BBB) crossing rate and specifically targeting capability. The as-prepared EVs@Dox/sgCD47/IL-9 was capable to deliver the Dox and gene editing platform into glioblastoma tissue and was assimilated by cancer cells efficiently, where the Dox induce the immunogenic cell death (ICD) of glioblastoma cells, the CRISPR-Cas9 platform down-regulated the expression of CD47 and the over-expression system up-regulated the expression of IL-9, both enhanced the immunity of biological system. Collectively, EVs@Dox/sgCD47/IL-9 demonstrated remarkable anti-tumor effects and reshaped the immunosuppressive tumor microenvironment that polarized the tumor-associated macrophages and activated CD8+ T cells with good biosafety. Thus, it establishes a strong foundation for the development of more effective strategies against glioblastoma. [ABSTRACT FROM AUTHOR]

Comparative ecophysiologists strive to understand physiological problems in non-model organisms, but molecular tools such as RNA interference (RNAi) are under-used in our field. Here, we provide a framework for invertebrate ecophysiologists to use RNAi to answer questions focused on physiological processes, rather than as a tool to investigate gene function. We specifically focus on non-model invertebrates, in which the use of other genetic tools (e.g. , genetic knockout lines) is less likely. We argue that because RNAi elicits a temporary manipulation of gene expression, and resources to carry out RNAi are technically and financially accessible, it is an effective tool for invertebrate ecophysiologists. We cover the terminology and basic mechanisms of RNA interference as an accessible introduction for "non-molecular" physiologists, include a suggested workflow for identifying RNAi gene targets and validating biologically relevant gene knockdowns, and present a hypothesis-testing framework for using RNAi to answer common questions in the realm of invertebrate ecophysiology. This review encourages invertebrate ecophysiologists to use these tools and workflows to explore physiological processes and bridge genotypes to phenotypes in their animal(s) of interest. [Display omitted] • RNAi is an accessible genetic tool that is underused in invertebrate physiology. • The RNAi target should be chosen to disrupt a physiological process of interest. • To appropriately interpret RNAi experiments, validation of the RNAi is required. • Careful timing of RNAi can provide strong insight into physiological processes. [ABSTRACT FROM AUTHOR]

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24–36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes’ integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum.

Background: The marine diatom Phaeodactylum tricornutum is a commercially viable species due to its bioactive substances and lipid productivity. Increasing attention has been paid to the isolation or genetic modification of species or strains with a rapid growth rate and large quantities of lipids. Furthermore, contamination of microzooplankton has been one of the major constraints in P. tricornutum large-scale cultivation, which adversely affects growth and greatly impedes the course of biomass production industrialization. Results: Here, based on our previous transcriptomics of P. tricornutum, we found a novel gene (ID: 7202015, hereafter called Pt2015) which affects morphotype of P. tricornutum. Pt2015 protein is located in the plastid, which is highly homologous to part of the sequences of exosome component. The morphotype of the Pt2015 knockout strain (termed 2015KO) using CRISPR/Cas9 method is fusiform, but the Pt2015 overexpression strain (termed oeT) demonstrates a majority triradiate morphotype (approximately 95%) which is stable and has been cultured for more than 200 generations. In addition, the oeT strain demonstrated a similar growth rate to the WT and simultaneously accumulated larger lipids droplets that increased by approximately 30% compared to that of the WT. More importantly, the grazing rate of the amoebae cultured in the oeT strain significantly decreased in comparison with that cultured in WT, suggesting that the oeT can effectively avoid being eaten by microzooplankton. Conclusions: Therefore, the oeT strain not only improves our understanding of morphotype conversion in diatoms but also demonstrates potential applications for large-scale cultivation of P. tricornutum. [ABSTRACT FROM AUTHOR]

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

Hepatocyte transplantation is an effective treatment for end-stage liver disease. However, due to the limited supply of human hepatocytes, porcine hepatocytes have garnered attention as a potential alternative source. Nonetheless, traditional primary porcine hepatocytes exhibit certain limitations in function maintenance and in vitro proliferation. This study has discovered that by using histone deacetylase inhibitors (HDACi), primary porcine hepatocytes can be successfully reprogrammed into liver progenitor cells with high proliferative potential. This method enables porcine hepatocytes to proliferate over an extended period in vitro and exhibit increased susceptibility in lentivirus-mediated gene modification. These liver progenitor cells can readily differentiate into mature hepatocytes and, upon microencapsulation transplantation into mice with acute liver failure, significantly improve the survival rate. This research provides new possibilities for the application of porcine hepatocytes in the treatment of end-stage liver disease. [Display omitted] • VPA can promote the long-term in vitro expansion of porcine hepatocytes by reprogramming PPHs into HPCs. • VPA-iHPCs showed high susceptibility to lentivirus-mediated gene modification. • VPA-iMHs showed mature liver function. • Microencapsulated VPA-iMHs can rescute acute liver failure mice. [ABSTRACT FROM AUTHOR]

Genetic modification provides an invaluable molecular tool to dissect the biology and pathogenesis of pathogens. However, no report is available about the genetic modification of Babesia duncani , a pathogen responsible for human babesiosis that is widespread in North America, suggesting the necessity to develop a genetic manipulation method to improve the strategies for studying and understanding the biology of protozoan pathogens. The establishment of a genetic modification method requires promoters, selectable markers, and reporter genes. Here, the double-copy gene elongation factor-1α (ef-1α) and its promoters were amplified by conventional PCR and confirmed by sequencing. We established a transient transfection system by using the ef-1αB promoter and the reporter gene mCherry and achieved stable transfection through homologous recombination to integrate the selection marker hDHFR-eGFP into the parasite genome. The potential of this genetic modification method was tested by knocking out the thioredoxin peroxidase-1 (TPX-1) gene, and under the drug pressure of 5 nM WR99210, 96.3% of the parasites were observed to express green fluorescence protein (eGFP) by flow cytometry at day 7 post-transfection. Additionally, the clone line of the TPX-1 knockout parasite was successfully obtained by the limiting dilution method. This study provided a transfection method for B. duncani , which may facilitate gene function research and vaccine development of B. duncani. [ABSTRACT FROM AUTHOR]

Precisely deciphering the cellular plasticity in vivo is essential in understanding many key biological processes. Site-specific recombinases are genetic tools used for in vivo lineage tracing and gene manipulation. Conventional Cre- loxP , Dre- rox , and Flp- frt technologies form the orthogonal recombination systems that can also be used in combination to increase the precision. As such, more than one marker gene can be targeted for lineage tracing, studying cellular heterogeneity, recording cellular activities, or even genome editing. Their combinatory use has recently resolved some controversies in defining cellular fate plasticity. Focusing on cell fate studies, we introduce the design principles of orthogonal recombinases-based strategies, describe some working examples in resolving cell fate-related controversies, and discuss some of their technical strengths and limits. Orthogonal recombinase systems are superior to conventional single-recombinase for studying in vivo cell fate, as they employ multiple markers to increase the resolution and precision of cell fate mapping results. Orthogonal recombinase systems enable genetic labeling of the intersect or subtractive parts of multiple cell populations, which helps clarify some controversial issues in cell origins and fate plasticity. Transitional transcriptional activation could be genetically recorded by orthogonal recombinase systems that could continuously capture some brief yet biologically important cellular events or states. Orthogonal recombinase systems have broad applications in cell lineage tracing and gene manipulation in fields of developmental biology, neuroscience, cardiovascular biology, oncology, and regenerative medicine. [ABSTRACT FROM AUTHOR]

Genetic modification provides an invaluable molecular tool to dissect the biology and pathogenesis of pathogens. However, no report is available about the genetic modification of Babesia duncani, a pathogen responsible for human babesiosis that is widespread in North America, suggesting the necessity to develop a genetic manipulation method to improve the strategies for studying and understanding the biology of protozoan pathogens. The establishment of a genetic modification method requires promoters, selectable markers, and reporter genes. Here, the double-copy gene elongation factor-1α (ef-1α) and its promoters were amplified by conventional PCR and confirmed by sequencing. We established a transient transfection system by using the ef-1αB promoter and the reporter gene mCherry and achieved stable transfection through homologous recombination to integrate the selection marker hDHFR-eGFP into the parasite genome. The potential of this genetic modification method was tested by knocking out the thioredoxin peroxidase-1 (TPX-1) gene, and under the drug pressure of 5 nM WR99210, 96.3% of the parasites were observed to express green fluorescence protein (eGFP) by flow cytometry at day 7 post-transfection. Additionally, the clone line of the TPX-1 knockout parasite was successfully obtained by the limiting dilution method. This study provided a transfection method for B. duncani, which may facilitate gene function research and vaccine development of B. duncani.

The quality of vegetables is facing new demands in terms of diversity and nutritional health. Given the improvements in living standards and the quality of consumed products, consumers are looking for vegetable products that maintain their nutrition, taste, and visual qualities. These requirements are directing scientists to focus on vegetable quality in breeding research. Thus, in recent years, research on vegetable quality has been widely carried out, and many applications have been developed via gene manipulation. In general, vegetable quality traits can be divided into three parts. First, commodity quality, which is most related to the commerciality of plants, refers to the appearance of the product. The second is flavor quality, which usually represents the texture and flavor of vegetables. Third, nutritional quality mainly refers to the contents of nutrients and health ingredients such as soluble solids (sugar), vitamin C, and minerals needed by humans. With biotechnological development, researchers can use gene manipulation technologies, such as molecular markers, transgenes and gene editing to improve the quality of vegetables. This review attempts to summarize recent studies on major vegetable crops species, with Brassicaceae, Solanaceae , and Cucurbitaceae as examples, to analyze the present situation of vegetable quality with the development of modern agriculture. [ABSTRACT FROM AUTHOR]

Over the last years, the CRISPR-Cas9 system has developed into the de facto standard gene modification technique in exploratory research. However, owing to its ease of usage and efficiency, CRISPR-Cas9 has also increasingly been exploited in the food industry while several applications have been reported for the brewing sector. Based on these developments, the present review aims at providing an overview of both the molecular background and function of the CRISPR-Cas9 system. This is followed by application examples of interest to the brewer. In the last part, the EU regulations and current scholarly discussion on genetically modified organisms are briefly summarized. [ABSTRACT FROM AUTHOR]

Abstract Background Gene editing is key for elucidating gene function. Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. However, cumbersome cloning and limited efficiency of negative selection often make this method slower than other methods such as recombineering. Results Here, we established a time-effective variant of consecutive single-crossovers. This method exploits rapid plasmid construction using Gibson assembly, a convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector resolution. We used this method to generate in-frame deletions, insertions and point mutations in Salmonella enterica with limited hands-on time. Adapted versions enabled efficient gene editing also in Pseudomonas aeruginosa and multi-drug resistant (MDR) Escherichia coli clinical isolates. Conclusions Our method is time-effective and allows facile manipulation of multiple bacterial species including MDR clinical isolates. We anticipate that this method might be broadly applicable to additional bacterial species, including those for which recombineering has been difficult to implement.

Abstract Background Crocus sativus is a recalcitrant plant for genetic transformation and genetic improvement, largely due to difficulties in Agrobacterium mediated transformation and vegetative reproduction. Effective genome editing requires proficient callus production and an efficient method to deliver Cas9 and sgRNAs into the plant. Here, we demonstrate Agrobacterium-mediated transformation of saffron. Further, we developed a CRISPR-Cas9 based system in this plant, for efficient gene knockout or edits in future. Results Efficient callus production and regeneration confers important benefits in developing competent transformation system in plants. More than 70% multiplication rate of callus initiation was achieved from corm slices of saffron subjected to a two-step sterilization procedure and grown on complete MS medium supplemented with 2,4-D (0.5 mg/L), BAP (1 mg/L), IAA (1 mg/L), photoperiod of 16/8 h and 45% relative humidity at 20 ± 2 °C. In vitro cormlet generation was accomplished in 8 weeks by using mature somatic embryos on MS medium supplemented with TDZ (0.5 mg/L) + IAA (1 mg/L) + Activated charcoal (0.1 g/L) at 15 ± 2 °C. The attempt of using Agrobacterium-mediated transformation resulted in successful integration of the binary vector into the somatic embryos of saffron with a transformation efficiency of 4%. PCR and Southern blot analysis confirmed the integration of Cas9 into saffron. Conclusion The protocol for callus production, somatic embryogenesis and regeneration was standardised. Successful demonstration of integrated Cas9 in this study constitutes first step in developing strategies for genetic manipulation of saffron, which has so far been considered recalcitrant. Furthering the development of this technology holds significant potential for advancing genetic research in saffron by integrating multigene targeting and/or use of recyclable cassettes.

Objective Lactic acid bacteria (LAB) synthesize valuable industrial and pharmaceutical important biopolymers, such as Alternan, by expressing glycosyltransferase enzymes by utilizing extracellular sucrose. In this study, due to the importance of such versatile biopolymers in the industry and medicine, a gene encoding an Altranansucrase (Asr) was introduced to sugar beet plants. Materials and methods A gene encoding the Asr gene was isolated from Leuconostoc mesenteroides bacterium and introduced to sugar beet plants by using Agrobacterium-mediated transformation. Molecular techniques were used to analyse transgenic plants and sugar content of sugar beet lines. Results Out of 131 transformed explants, only three transgenic plants were produced, showing a transformation efficiency of 2.3%. The Asr gene integration in transgenic plants genome and expression were confirmed by specific PCR and semi-quantitative RT-PCR analysis, respectively. Sugar analysis of sugar beet transgenic plants showed that control plants with a 19.6% brix value (Sucrose) had more sucrose than transgenic plants with an average brix value of 14.4%. Brix in transgenic plants was lower than that of control plants. The amount of sugar (sucrose) in the transgenic asr-expressing plants reduced by 36.1% relative to the untransformed control plants. Conclusions The results of this study showed that Altrenansucrase can convert substantial amount of sugar beet sucrose into Alternan biopolymer, producing 36.6 mg/g FW alternan biopolymer with pharmaceutical and industrial applications.

The quality of vegetables is facing new demands in terms of diversity and nutritional health. Given the improvements in living standards and the quality of consumed products, consumers are looking for vegetable products that maintain their nutrition, taste, and visual qualities. These requirements are directing scientists to focus on vegetable quality in breeding research. Thus, in recent years, research on vegetable quality has been widely carried out, and many applications have been developed via gene manipulation. In general, vegetable quality traits can be divided into three parts. First, commodity quality, which is most related to the commerciality of plants, refers to the appearance of the product. The second is flavor quality, which usually represents the texture and flavor of vegetables. Third, nutritional quality mainly refers to the contents of nutrients and health ingredients such as soluble solids (sugar), vitamin C, and minerals needed by humans. With biotechnological development, researchers can use gene manipulation technologies, such as molecular markers, transgenes and gene editing to improve the quality of vegetables. This review attempts to summarize recent studies on major vegetable crops species, with Brassicaceae, Solanaceae, and Cucurbitaceae as examples, to analyze the present situation of vegetable quality with the development of modern agriculture.

Microbial electrocatalysis reckons on microbes as catalysts for reactions occurring at electrodes. Microbial fuel cells and microbial electrolysis cells are well-known in this context; both prefer the oxidation of organic and inorganic matter for producing electricity. Notably, the synthesis of high energy-density chemicals (fuels) or their precursors by microorganisms using biocathode to yield electrical energy is called Microbial Electrosynthesis (MES), giving an exceptionally appealing novel way for producing beneficial products from electricity and wastewater. This review accentuates the concept, importance and opportunities of MES, as an emerging discipline at the nexus of microbiology and electrochemistry. Production of organic compounds from MES is considered as an effective technique for the generation of various beneficial reduced end-products (like acetate and butyrate) as well as in reducing the load of CO2 from the atmosphere to mitigate the harmful effect of greenhouse gases in global warming. Although MES is still an emerging technology, this method is not thoroughly known. The authors have focused on MES, as it is the next transformative, viable alternative technology to decrease the repercussions of surplus carbon dioxide in the environment along with conserving energy. [ABSTRACT FROM AUTHOR]

The development of new techniques to create gene knockouts and knock-ins is essential for successful investigation of gene functions and elucidation of the causes of diseases and their associated fundamental cellular processes. In the biomedical model organism Dictyostelium discoideum, the methodology for gene targeting with homologous recombination to generate mutants is well-established. Recently, we have applied CRISPR/Cas9-mediated approaches in Dictyostelium, allowing the rapid generation of mutants by transiently expressing sgRNA and Cas9 using an all-in-one vector. CRISPR/Cas9 techniques not only provide an alternative to homologous recombination-based gene knockouts but also enable the creation of mutants that were technically unfeasible previously. Herein, we provide a detailed protocol for the CRISPR/Cas9-based method in Dictyostelium. We also describe new tools, including double knockouts using a single CRISPR vector, drug-inducible knockouts, and gene knockdown using CRISPR interference (CRISPRi). We demonstrate the use of these tools for some candidate genes. Our data indicate that more suitable mutants can be rapidly generated using CRISPR/Cas9-based techniques to study gene function in Dictyostelium.

Background: Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in Leishmania parasites. Methods: U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gp63 gene and cloned in pX-leish. Results: The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. Conclusion: This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future.

The plant growth promoting rhizobacteria (PGPR) has multifarious benefits to serve sustainable agriculture. Different consortia of microbes are currently being in use to increase yield of different commercial crops including wheat, canola, lettuce, tomato, banana, maize, chickpea and brinjal. Introduction of PGPR strains can help restore the soil health and prevent the ill effects of fertilizers. However, complete replacement of chemical fertilizers need engineered microbes with enhanced PGPR potential. Researchers are working seamlessly to engineer microbes with augmented indole acetic acid production, disease resistance, chitinases activity, nitrogen fixation and phosphorus solubilisation potential. Advances in gene editing tools and omics technologies have eased the process of gene manipulation in bacteria allowing non-PGPR strains to work as PGPR inoculants in rhizosphere. In this review, we have discussed microbe-based rhizospheric engineering approaches for the manipulation of different gene of interest for improving the PGPR potential of PGPR and non-PGPR strains. This review is fundamental in providing first-hand information on different success stories of genetic manipulations of PGPR strains done in recent years and provides understanding of the process in shaping future rhizosphere microbiome. [ABSTRACT FROM AUTHOR]

Human babesiosis is a tick-borne disease caused by Babesia pathogens. The disease, which presents with malaria-like symptoms, can be life-threatening, especially in individuals with weakened immune systems and the elderly. The worldwide prevalence of human babesiosis has been gradually rising, prompting alarm among public health experts. In other pathogens, genetic techniques have proven to be valuable tools for conducting functional studies to understand the importance of specific genes in development and pathogenesis as well as to validate novel cellular targets for drug discovery. Genetic manipulation methods have been established for several non-human Babesia and Theileria species and, more recently, have begun to be developed for human Babesia parasites. We have previously reported the development of a method for genetic manipulation of the human pathogen Babesia duncani . This method is based on positive selection using the hDHFR gene as a selectable marker, whose expression is regulated by the ef-1aB promoter, along with homology regions that facilitate integration into the gene of interest through homologous recombination. Herein, we provide a detailed description of the steps needed to implement this strategy in B. duncani to study gene function. It is anticipated that the implementation of this method will significantly improve our understanding of babesiosis and facilitate the development of novel and more effective therapeutic strategies for the treatment of human babesiosis. Key features This protocol provides an effective means of transfection of B. duncani , enabling genetic manipulation and editing to gain further insights into its biology and pathogenesis. The protocol outlined here for the electroporation of B. duncani represents an advancement over previous methods used for B. bovis [1]. Improvements include higher volume of culture used during the electroporation step and an enhancement in the number of electroporation pulses. These modifications likely enhance the efficiency of gene editing in B. duncani , allowing for quicker and more effective selection of transgenic parasites., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Abstract Background Human infertility has become a serious and social issue all over the world, especially in developed countries. Numerous types of assisted reproductive technology have been developed and are widely used to treat infertility. However, pregnancy outcomes require further improvement. It is essential to understand the cross‐talk between the uterus (mother) and the embryo (fetus) in pregnancy, which is a very complicated event. Methods The mammalian uterus requires many physiological and morphological changes for pregnancy‐associated events, including implantation, decidualization, placentation, and parturition, to occur. Here is discussed recent advances in the knowledge of the molecular mechanisms underlying these reproductive events — in particular, embryonic implantation and decidualization — based on original and review articles. Main findings (Results) In mice, embryonic implantation and decidualization are regulated by two steroid hormones: estrogen and progesterone. Along with these hormones, cytokines, cell‐cycle regulators, growth factors, and transcription factors have essential roles in implantation and decidualization in mice. Conclusion Recent studies using the gene manipulation of mice have given considerable insight into the molecular mechanisms underlying embryonic implantation and decidualization. However, as most of the findings are based on mice, comparative research using different mammalian species will be useful for a better understanding of the species‐dependent differences that are associated with reproductive events, including embryonic implantation.

Background: Gene editing is key for elucidating gene function. Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. However, cumbersome cloning and limited efficiency of negative selection often make this method slower than other methods such as recombineering. Results: Here, we established a time-effective variant of consecutive single-crossovers. This method exploits rapid plasmid construction using Gibson assembly, a convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector resolution. We used this method to generate in-frame deletions, insertions and point mutations in Salmonella enterica with limited hands-on time. Adapted versions enabled efficient gene editing also in Pseudomonas aeruginosa and multi-drug resistant (MDR) Escherichia coli clinical isolates. Conclusions: Our method is time-effective and allows facile manipulation of multiple bacterial species including MDR clinical isolates. We anticipate that this method might be broadly applicable to additional bacterial species, including those for which recombineering has been difficult to implement. [ABSTRACT FROM AUTHOR]

Background: Crocus sativus is a recalcitrant plant for genetic transformation and genetic improvement, largely due to difficulties in Agrobacterium mediated transformation and vegetative reproduction. Effective genome editing requires proficient callus production and an efficient method to deliver Cas9 and sgRNAs into the plant. Here, we demonstrate Agrobacterium-mediated transformation of saffron. Further, we developed a CRISPR-Cas9 based system in this plant, for efficient gene knockout or edits in future. Results: Efficient callus production and regeneration confers important benefits in developing competent transformation system in plants. More than 70% multiplication rate of callus initiation was achieved from corm slices of saffron subjected to a two-step sterilization procedure and grown on complete MS medium supplemented with 2,4-D (0.5 mg/L), BAP (1 mg/L), IAA (1 mg/L), photoperiod of 16/8 h and 45% relative humidity at 20 ± 2 °C. In vitro cormlet generation was accomplished in 8 weeks by using mature somatic embryos on MS medium supplemented with TDZ (0.5 mg/L) + IAA (1 mg/L) + Activated charcoal (0.1 g/L) at 15 ± 2 °C. The attempt of using Agrobacterium-mediated transformation resulted in successful integration of the binary vector into the somatic embryos of saffron with a transformation efficiency of 4%. PCR and Southern blot analysis confirmed the integration of Cas9 into saffron. Conclusion: The protocol for callus production, somatic embryogenesis and regeneration was standardised. Successful demonstration of integrated Cas9 in this study constitutes first step in developing strategies for genetic manipulation of saffron, which has so far been considered recalcitrant. Furthering the development of this technology holds significant potential for advancing genetic research in saffron by integrating multigene targeting and/or use of recyclable cassettes. [ABSTRACT FROM AUTHOR]

Genetic manipulation in mammals has progressed rapidly in the past decade with the advent of CRISPR-Cas gene editing tools, promising profound impacts on the understanding of human development, health and disease. However, many years of research in divergent fields of experimental embryology, genetics, reproduction, molecular biology and transgenic technology laid the groundwork and have played critical roles for this progress. This article details various threads of research and the central role of the laboratory mouse that came together in reaching this point, all from the perspective of a scientist whose research was deeply immersed in the field., (Copyright © 2024 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Epigenetic modification, as typified by cytosine methylation, is a key aspect of gene regulation that affects many biological processes. However, the biological roles of individual methylated cytosines are poorly understood. Sequence-specific DNA recognition tools can be used to investigate the roles of individual instances of DNA methylation. Transcription activator-like effectors (TALEs), which are DNA-binding proteins, are promising candidate tools with designable sequence specificity and sensitivity to DNA methylation. In this review, we describe the bases of DNA recognition of TALEs, including methylated cytosine recognition, and the applications of TALEs for the study of methylated DNA. In addition, we discuss TALE-based epigenome editing and oxidized methylated cytosine recognition. Unlabelled Image [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI), a prominent health issue, represents a substantial portion of the global health care burden. Stem cell-based therapies provide novel solutions for SCI treatment, yet obstacles remain in the form of low survival rate, uncontrolled differentiation, and functional recovery. The application of engineered biomaterials in stem cell therapy provides a physicochemical microenvironment that mimics the stem cell niche, facilitating self-renewal, stem cell differentiation, and tissue reorganization. Nonetheless, external microenvironment support is inadequate, and some obstacles persist, for example, limited sources, gradual aging, and immunogenicity of stem cells. Targeted stem cell gene manipulation could eliminate many of these drawbacks, allowing safer, more effective use under regulation of intrinsic mechanisms. Additionally, through genetic labeling of stem cells, their role in tissue engineering may be elucidated. Therefore, combining stem cell therapy, materials science, and genetic modification technologies may shed light on SCI treatment. Herein, recent advances and advantages of biomaterials and gene manipulation, especially with respect to stem cell-based therapies, are highlighted, and their joint performance in SCI is evaluated. Current technological limitations and perspectives on future directions are then discussed. Although this combination is still in the early stages of development, it is highly likely to substantially contribute to stem cell-based therapies in the foreseeable future. [ABSTRACT FROM AUTHOR]

Background: Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in Leishmania parasites. Methods: U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gp63 gene and cloned in pX-leish. Results: The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. Conclusion: This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future. [ABSTRACT FROM AUTHOR]

Skeletal muscle mass differs greatly in mice and humans and this is partially inherited. To identify muscle hypertrophy candidate genes we conducted a systematic review to identify genes whose experimental loss or gain-of-function results in significant skeletal muscle hypertrophy in mice. We found 47 genes that meet our search criteria and cause muscle hypertrophy after gene manipulation. They are from high to small effect size: Ski, Fst, Acvr2b, Akt1, Mstn, Klf10, Rheb, Igf1, Pappa, Ppard, Ikbkb, Fstl3, Atgr1a, Ucn3, Mcu, Junb, Ncor1, Gprasp1, Grb10, Mmp9, Dgkz, Ppargc1a (specifically the Ppargc1a4 isoform), Smad4, Ltbp4, Bmpr1a, Crtc2, Xiap, Dgat1, Thra, Adrb2, Asb15, Cast, Eif2b5, Bdkrb2, Tpt1, Nr3c1, Nr4a1, Gnas, Pld1, Crym, Camkk1, Yap1, Inhba, Tp53inp2, Inhbb, Nol3, Esr1. Knock out, knock down, overexpression or a higher activity of these genes causes overall muscle hypertrophy as measured by an increased muscle weight or cross sectional area. The mean effect sizes range from 5 to 345% depending on the manipulated gene as well as the muscle size variable and muscle investigated. Bioinformatical analyses reveal that Asb15, Klf10, Tpt1 are most highly expressed hypertrophy genes in human skeletal muscle when compared to other tissues. Many of the muscle hypertrophy-regulating genes are involved in transcription and ubiquitination. Especially genes belonging to three signaling pathways are able to induce hypertrophy: (a) Igf1-Akt-mTOR pathway, (b) myostatin-Smad signaling, and (c) the angiotensin-bradykinin signaling pathway. The expression of several muscle hypertrophy-inducing genes and the phosphorylation of their protein products changes after human resistance and high intensity exercise, in maximally stimulated mouse muscle or in overloaded mouse plantaris.