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Background/aim: Griscelli Syndrome Type 2 (GS-2) is a rare, inherited immune deficiency caused by a mutation in the RAB27A gene. The current treatment consists of hematopoietic stem cell transplantation, but a lack of suitable donors warrants the development of alternative treatment strategies, including gene therapy. The development of mutation-specific clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing technology has opened the way for custom-designed gene correction of patientderived stem cells. In this study, we aimed to custom design CRISPR/Cas9 constructs and test their efficiency on homology-directed repair (HDR) on the correction of exon 3 and exon 7 mutations in the RAB27A gene of GS-2 patient-derived mesenchymal stem cells (MSCs) and induced pluripotent stem cells. Materials and methods: We assessed RAB27A gene and protein expression using qRT-PCR, Western Blot, and immune fluorescence in GS-2 patient-derived MSCs and induced pluripotent stem cells (iPSCs). Guide RNAs (gRNAs) and donor DNAs were designed based on patient mutations in exon 3 and exon 7 using the CHOPCHOP online tool and transfected into GS-2 MSCs and iPSCs by electroporation. The cells were cultured for 2 days and then used for mutation analysis using DNA sequencing. Results: MSCs and iPSCs from the GS-2 patients lacked RAB27A gene and protein expression. After gRNA and donor DNAs were designed and optimized, we found HDR efficiency with gRNA3.3 (10% efficiency) and gRNA7.3 (27% efficiency) for MSCs but lower efficiency in iPSCs (<5%). However, transfection of both MSCs and iPSCs resulted in massive cell death, loss of colony formation, and spontaneous differentiation. Conclusion: The use of CRISPR/Cas9 to genetically correct MSCs and iPSCs from GS-2 patients with different mutations through HDR is feasible but requires optimization of the procedure to reduce cell death and improve stem cell function before clinical application. [ABSTRACT FROM AUTHOR]

Perilla [ Perilla frutescens (L.) var frutescens ] is a traditional oil crop in Asia, recognized for its seeds abundant in α-linolenic acid (18:3), a key omega-3 fatty acid known for its health benefits. Despite the known nutritional value, the reason behind the higher 18:3 content in tetraploid perilla seeds remained unexplored. Gamma irradiation yielded mutants with altered seed fatty acid composition. Among the mutants, DY-46-5 showed a 27% increase in 18:2 due to the 4-bp deletion of PfrFAD3b , and NC-65-12 displayed a 16% increase in 18:2 due to the loss of function of PfrFAD3 a through a large deletion. Knocking out both copies of FATTY ACID DESATURASE3 (PfrFAD3a and PfrFAD3b) simultaneously using CRISPR/Cas9 resulted in an increase in 18:2 by up to 75% and a decrease in 18:3 to as low as 0.3% in seeds, emphasizing the pivotal roles of both genes in 18:3 synthesis in tetraploid perilla. Furthermore, diploid Perilla citriodora , the progenitor of cultivated tetraploid perilla, harbors only PfrFAD3b , with a fatty acid analysis revealing lower 18:3 levels than tetraploid perilla. In conclusion, the enhanced 18:3 content in cultivated tetraploid perilla seeds can be attributed to the acquisition of two FAD3 copies through hybridization with wild-type diploid perilla. [ABSTRACT FROM AUTHOR]

Objective: To assess the impact of the IFIT3/TBK1 signalling pathway in activating plasmacytoid dendritic cells (pDCs) and its role in the development of SSc. Methods: Utilized single‐cell RNA sequencing (scRNA‐seq) and high‐throughput transcriptome RNA sequencing to reveal the differential abundance of pDCs and the role of the key gene IFIT3 in SSc. Conducted in vitro cell experiments to evaluate the effect of IFIT3/TBK1 signalling pathway intervention on pDC activation cytokine release and fibroblast function. Constructed an IFIT3−/− mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) gene editing to assess the potential benefits of intervening in the IFIT3/TBK1 signalling pathway on skin and lung fibrosis in the SSc mouse model. Results: The IFIT3/TBK1 signalling pathway plays a crucial role in activating pDCs, with IFIT3 acting as an upstream regulator of TBK1. Intervention in the IFIT3/TBK1 signalling pathway can inhibit pDC activation cytokine release and impact fibroblast function. The IFIT3−/− mouse model shows potential benefits of targeting the IFIT3/TBK1 signalling pathway in reducing skin and lung fibrosis in the SSc mouse model. Conclusion: This study provides new insights into potential therapeutic targets for SSc, highlighting the critical role of the IFIT3/TBK1 signalling pathway in SSc development. Highlights: This study elucidates the pivotal role of plasmacytoid dendritic cells (pDCs) in systemic sclerosis (SSc).This study identified the key regulatory gene involved in systemic sclerosis (SSc) as IFIT3.This study has found that IFIT3 functions as an upstream regulatory factor, activating TBK1.This study provides Evidence of the regulatory effects of the IFIT3/TBK1 pathway on plasmacytoid dendritic cells (pDCs).This study validated the therapeutic potential using the IFIT3−/− mouse model. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a gene-editing technology. Nanoparticle delivery systems have attracted attention because of the limitations of conventional viral vectors. In this review, we assess the efficiency of various nanoparticles, including lipid-based, polymer-based, inorganic, and extracellular vesicle-based systems, as non-viral vectors for CRISPR/Cas9 delivery. We discuss their advantages, limitations, and current challenges. By summarizing recent advancements and highlighting key strategies, this review aims to provide a comprehensive overview of the role of non-viral delivery systems in advancing CRISPR/Cas9 technology for clinical applications and gene therapy. [ABSTRACT FROM AUTHOR]

Previous research has highlighted significant phenotypic discrepancies between knockout and knockdown approaches in zebrafish, raising concerns about the reliability of these methods. However, our study suggests that these differences are not as pronounced as was once believed. By carefully examining the roles of maternal and zygotic gene contributions, we demonstrate that these factors significantly influence phenotypic outcomes, often accounting for the observed discrepancies. Our findings emphasize that morpholinos, despite their potential off-target effects, can be effective tools when used with rigorous controls. We introduce the concept of graded maternal contribution, which explains how the uneven distribution of maternal mRNA and proteins during gametogenesis impacts phenotypic variability. Our research categorizes genes into three types—susceptible, immune, and "Schrödinger" (conditional)—based on their phenotypic expression and interaction with genetic compensation mechanisms. This distinction provides new insights into the paradoxical outcomes observed in genetic studies. Ultimately, our work underscores the importance of considering both maternal and zygotic contributions, alongside rigorous experimental controls, to accurately interpret gene function and the mechanisms underlying disease. This study advocates for the continued use of morpholinos in conjunction with advanced genetic tools like CRISPR/Cas9, stressing the need for a meticulous experimental design to optimize the utility of zebrafish in genetic research and therapeutic development. [ABSTRACT FROM AUTHOR]

Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae) is one of the causal agents of wheat viral disease, which severely impacts wheat production in most wheat-growing regions in the world. Currently, there is little information about natural resistance against WDV in common wheat germplasms. CRISPR/Cas9 technology is being utilized to manufacture transgenic plants resistant to different diseases. In the present study, we used the CRISPR/Cas9 system targeting overlapping regions of coat protein (CP) and movement protein (MP) (referred to as CP/MP) or large intergenic region (LIR) in the wheat variety 'Fielder' to develop resistance against WDV. WDV-inoculated T1 progenies expressing Cas9 and sgRNA for CP/MP and LIR showed complete resistance against WDV and no accumulation of viral DNA compared with control plants. Mutation analysis revealed that the CP/MP and LIR targeting sites have small indels in the corresponding Cas9-positive plants. Additionally, virus inhibition and indel mutations occurred in T2 homozygous lines. Together, our work gives efficient results of the engineering of CRISPR/Cas9-mediated WDV resistance in common wheat plants, and the specific sgRNAs identified in this study can be extended to utilize the CRISPR/Cas9 system to confer resistance to WDV in other cereal crops such as barley, oats, and rye. [ABSTRACT FROM AUTHOR]

Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia (Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species, the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyperosmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5′-GGAAA-3′) matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO-challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (P < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (P < 0.005). Furthermore, reductions of IMPA1.1 (37–49%) and MIPS (6–37%) mRNA abundance were observed in HO-challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO-induced activation of the MIB pathway. NEW & NOTEWORTHY: In our study, we use a multi-pronged synthetic biology approach to demonstrate that the fish homolog of the predominant mammalian osmotic stress transcription factor nuclear factor of activated T-cells (NFAT5) also contributes to the activation of hyperosmolality inducible genes in cells of extremely euryhaline fish. However, in addition to NFAT5 the presence of other strong osmotically inducible signaling mechanisms is required for full activation of osmoregulated tilapia genes. [ABSTRACT FROM AUTHOR]

Gene regulatory networks specify the gene expression patterns needed for traits to develop. Differences in these networks can result in phenotypic differences between organisms. Although loss-of-function genetic screens can identify genes necessary for trait formation, gain-of-function screens can overcome genetic redundancy and identify loci whose expression is sufficient to alter trait formation. Here, we leveraged transgenic lines from the Transgenic RNAi Project at Harvard Medical School to perform both gain- and loss-of-function CRISPR/Cas9 screens for abdominal pigmentation phenotypes. We identified measurable effects on pigmentation patterns in the Drosophila melanogaster abdomen for 21 of 55 transcription factors in gain-of-function experiments and 7 of 16 tested by loss-of-function experiments. These included well-characterized pigmentation genes, such as bab1 and dsx , and transcription factors that had no known role in pigmentation, such as slp2. Finally, this screen was partially conducted by undergraduate students in a Genetics Laboratory course during the spring semesters of 2021 and 2022. We found this screen to be a successful model for student engagement in research in an undergraduate laboratory course that can be readily adapted to evaluate the effect of hundreds of genes on many different Drosophila traits, with minimal resources. [ABSTRACT FROM AUTHOR]

Pest management based on CRISPR/Cas9-mediated site-specific mutations is an effective and environmentally safer strategy to suppress the pest population. However, the potential of this approach is yet to be tested on many important agricultural pests such as Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), a fit candidate for area-wide pest management. Therefore, in the present study, 2 spermatogenesis-related genes viz. Testis-specific zinc finger protein (topi) and Testis-specific serine protein kinase 1 (Tssk1) of B. dorsalis were edited to impart male sterility and its impact on further progeny. In this regard, topi and Tssk1 mutant populations deposited significantly fewer eggs per day (6.12 ± 0.36 and 3.60 ± 0.24, respectively) as compared to the control (11.16 ± 0.58 eggs per day). About the hatching rate, the above trend was observed, topi (44.51) and Tssk1 (30.04) as compared to the control (73.96). Furthermore, the total number of viable offspring for topi and Tssk1 populations decreased as a result of the cumulative progeny production ten days after the post-mating phase. It suggests that topi and Tssk1 from B. dorsalis could be potential targets for imparting male sterility in B. dorsalis. [ABSTRACT FROM AUTHOR]

Aquaculture, the world's fastest-growing food production sector, is critical for addressing food security concerns because of its potential to deliver high-quality, nutrient-rich supplies by 2050. This review assesses the effectiveness of CRISPR/Cas9 genome editing technology in enhancing desirable traits in fish species, including growth rates, muscle quality, disease resistance, pigmentation, and more. It also focuses on the potential effectiveness of the technology in allowing precise and targeted modifications of fish DNA to improve desirable characteristics. Many studies have reported successful applications of CRISPR/Cas9, such as knocking out reproductive genes to control reproduction and sex determination, enhancing feed conversion efficiency, and reducing off-target effects. Additionally, this technology has contributed to environmental sustainability by reducing nitrogen-rich waste and improving the nutritional composition of fish. However, the acceptance of CRISPR/Cas9 modified fish by the public and consumers is hindered by concerns regarding public perception, potential ecological impacts, and regulatory frameworks. To gain public approval and consumer confidence, clear communication about the editing process, as well as data on the safety and environmental considerations of genetically modified fish, are essential. This review paper discusses these challenges, provides possible solutions, and recommends future research on the integration of CRISPR/Cas9 into sustainable aquaculture practices, focusing on the responsible management of genetically modified fish to enable the creation of growth and disease-resistant strains. In conclusion, this review highlights the transformative potential of CRISPR/Cas9 technology in improving fish traits, while also considering the challenges and ethical considerations associated with sustainable and responsible practices in aquaculture. [ABSTRACT FROM AUTHOR]

The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation. [ABSTRACT FROM AUTHOR]

Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications. Synopsis: While off-target mutations have been carefully addressed, CRISPR/Cas9 mediated genome editing often causes harmful on-target chromosomal alterations. CasPlus combines Cas9 genome editing with an engineered phage T4 DNA polymerase and reduces the incidence of large deletions and chromosomal translocations. CasPlus can be applied to correct disease-causing mutations such as in Duchenne muscular dystrophy (DMD), but also for generating engineered T cells for cell therapy. CasPlus editing inhibits undesired on-target large deletions while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing. CasPlus efficiently corrects the frameshift mutations in DMD (exon 52 deletions) with fewer on-target DNA damage. CasPlus greatly reduces the frequency of on-target large deletions in mouse germline editing. CasPlus represses chromosomal translocation while maintaining gene disruption efficiency that is higher or comparable to Cas9 in multiplex gene-edited human primary T cells. CasPlus combines Cas9 genome editing with an engineered phage T4 DNA polymerase and reduces the incidence of large deletions and chromosomal translocations. [ABSTRACT FROM AUTHOR]

In this study, we established SH-SY5Y human neuroblastoma cells as an in vitro model to investigate whether oxidative stress and the nuclear erythroid-2 related factor 2 (Nrf2) signaling pathway are associated with 1-bromopropane (1-BP) -induced nerve cell injury. We identified that 1-BP exhibited neurotoxicity mainly through oxidant-based processes in SH-SY5Y cells, as reactive oxygen species, malondialdehyde levels, and 8-hydroxy-2' -deoxyguanosine significantly increased, while superoxide dismutase activity decreased. Furthermore, Nrf2 translocation from the cytosol to the nucleus was inhibited, as was downstream protein expression of the Nrf2-regulated genes HO-1 and Bcl-2. Activation of caspase-9 and −3 increased, and apoptosis was observed. Vitamin C alleviated 1-BP-induced apoptosis by decreasing oxidative stress and activating the Nrf2 signaling pathway. Knockdown of Nrf2 in SH-SY5Y cells increased 1-BP-induced reactive oxygen species production and cell apoptosis, and inhibited HO-1 and Bcl-2 protein expression, while overexpression of Nrf2 alleviated these processes. These findings suggest that 1-BP-induced oxidative stress and apoptosis in SH-SY5Y cells are associated with Nrf2 function inhibition. [ABSTRACT FROM AUTHOR]

• This review article highlights the challenge of limited genetic diversity in tomatoes, emphasizing the hurdles it poses to traditional breeding efforts. • The emergence of CRISPR/Cas9 genome editing is described as revolutionary, signaling a transformative shift in the efficiency of tomato breeding. • The variants of CRISPR/Cas9 technology in editing and characterizing a wide range of traits in tomatoes, highlighting its effectiveness in manipulating the tomato genome. • Several innovative techniques within the CRISPR/Cas technology, such as online tools for designing RNA, advanced cloning techniques like Golden Gate cloning, and various Cas9 variants and editing techniques are discussed. • Advancements in CRISPR/Cas technology, including specific constructs, efficient transformation methodologies, and DNA-free protoplast methods, all contributing to enhanced editing capabilities and efficiency in horticultural crops breeding are briefly discussed. The limited genetic diversity in tomatoes presents significant hurdles for traditional breeding efforts. However, the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein 9 (CRISPR/Cas9) genome editing has revolutionized the rate and efficiency of tomato breeding. CRISPR/Cas9 technology plays a crucial role in editing and thoroughly characterizing various traits in tomatoes. CRISPR/Cas9 has shown efficacy in transferring novel domestication traits between cultivated tomatoes and their wild relatives and vice versa. A number of notable developments in CRISPR/Cas technology include the utilization of online resources for multiplexing and designing single-guide RNA (sgRNA), as well as utilizing cutting-edge cloning methods such as GoldenBraid, BioBrick, and Golden Gate cloning technology. Furthermore, improved transformation methods, such as Agrobacterium -mediated methods, the development of CRISPR/Cas constructs, and the use of DNA-free protoplasts for Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) complexes, contribute to the advancement of gene editing capabilities. The toolkit for precise genome modification has also been expanded by the application of Cas9 variants, such as Cas9-NG/XNG-Cas9 and protospacer-adjacent motif (PAM) free Cas12a (Cpf1), together with techniques such as base/prime editing using Target- Activation-Induced Cytidine Deaminase (AID) technology and homologous recombination (HR)-based gene knock-in (HKI) mediated by geminivirus replicon. This review focuses on the significant advancements made in current research using CRISPR/Cas technology for fast and successful tomato breeding. [ABSTRACT FROM AUTHOR]

The innovations and progress in genome editing/new breeding technologies have revolutionized research in the field of functional genomics and crop improvement. This revolution has expanded the horizons of agricultural research, presenting fresh possibilities for creating novel plant varieties equipped with desired traits that can effectively combat the challenges posed by climate change. However, the regulation and social acceptance of genome-edited crops still remain as major barriers. Only a few countries considered the site-directed nuclease 1 (SDN1) approach-based genome-edited plants under less or no regulation. Hence, the present review aims to comprise information on the research work conducted using SDN1 in crops by various genome editing tools. It also elucidates the promising candidate genes that can be used for editing and has listed the studies on non-transgenic crops developed through SDN1 either by Agrobacterium-mediated transformation or by ribo nucleoprotein (RNP) complex. The review also hoards the existing regulatory landscape of genome editing and provides an overview of globally commercialized genome-edited crops. These compilations will enable confidence in researchers and policymakers, across the globe, to recognize the full potential of this technology and reconsider the regulatory aspects associated with genome-edited crops. Furthermore, this compilation serves as a valuable resource for researchers embarking on the development of customized non-transgenic crops through the utilization of SDN1. [ABSTRACT FROM AUTHOR]

LCP1 encodes L-plastin, an actin-bundling protein primarily expressed in hematopoietic cells. In mouse and fish models, LCP1 deficiency has been shown to result in hematologic and immune defects. This study aimed to determine the nature of a human inborn error of immunity resulting from a novel genetic variant of LCP1. We performed genetic, protein, and cellular analysis of PBMCs from a kindred with apparent autosomal dominant immune deficiency. We identified a candidate causal mutation in LCP1 , which we evaluated by engineering the orthologous mutation in mice and Jurkat cells. A splice-site variant in LCP1 segregated with lymphopenia, neutropenia, and thrombocytopenia. The splicing defect resulted in at least 2 aberrant transcripts, producing an in-frame deletion of 24 nucleotides, and a frameshift deletion of exon 8. Cellular analysis of the kindred revealed a proportionate reduction of T and B cells and a mild expansion of transitional B cells. Similarly, mice carrying the orthologous genetic variant exhibited the same in-frame aberrant transcript, reduced expression Lcp1 and gene dose-dependent leukopenia, mild thrombocytopenia, and lymphopenia, with a significant reduction of T-cell populations. Functional analysis revealed that LCP1c740-1G>A confers a defect in platelet development and function with aberrant spreading on collagen. Immunologic analysis revealed defective actin organization in T cells, reduced migration of PBMCs from patients, splenocytes from mutant mice, and a mutant Jurkat cell line in response to CXCL12; impaired germinal center B-cell expansion after immunization; and reduced cytokinesis during T cell proliferation. We describe a unique human hematopoietic defect affecting neutrophils, lymphocytes, and platelets arising from partial LCP1 deficiency. [ABSTRACT FROM AUTHOR]

Sheath blight, caused by the fungus Rhizoctonia solani, is a major problem that significantly impacts rice production and can lead to substantial yield losses. The disease has become increasingly problematic in recent years due to the widespread use of high-yielding semi-dwarf rice cultivars, dense planting, and heavy application of nitrogenous fertilizers. The disease has become more challenging to manage due to its diverse host range and the lack of resistant cultivars. Despite utilizing traditional methods, the problem persists without a satisfactory solution. Therefore, modern approaches, including advanced breeding, transgenic methods, genome editing using CRISPR/Cas9 technology, and nanotechnological interventions, are being explored to develop rice plants resistant to sheath blight disease. This review primarily focuses on these recent advancements in combating the sheath blight disease. [ABSTRACT FROM AUTHOR]

Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) is the key enzyme in the biosynthetic pathway of chlorogenic acid. A homologous gene of NtHQT from Nicotiana tabacum was cloned and named NtHQT2. Amino acid sequence alignment and phylogenetic analysis revealed that NtHQT2 possessed the conserved HTLSD and DEFEG motifs of HQT protein and belonged to the same branch with HQTs from tobacco and other Solanaceae plants in terms of evolutionary relationships. By analyzing the expression patterns of NtHQT2 gene in different tissues of tobacco by qRT-PCR analysis, it was found that NtHQT2 gene was expressed in the root, stalk, leaf, axillary bud, and flower with higher expression levels observed in the leaves. CRISPR/Cas9 gene editing technology was used to perform targeted knockout of NtHQT2 gene, and three types of T0 generation gene-edited plants with NtHQT2 translation error were obtained. By quantifying the chlorogenic acid content in the leaves of homozygous T2 NtHQT2 gene-edited plants, it was observed that the edited plants exhibited a significant decrease in chlorogenic acid content compared to the control, indicating that NtHQT2 gene positively regulates the synthesis of chlorogenic acid. [ABSTRACT FROM AUTHOR]

SUMMARY: Many viral diseases affect the poultry industry worldwide and pose a devastating economic impact in terms of morbidity and mortality. Generally, there is no treatment for these viral infections, but vaccines are used to control these diseases as prophylaxis. The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein9), a novel and cost-effective gene editing technique, has recently opened new horizons to find new treatments for viral infections by altering the genome of the host or virus. Moreover, this approach can be used to characterise host-virus affinity, elucidate the role of the viral genome, develop recombinant viral vaccines, modify the viral genome to discover antiviral therapies, and disease resistance. The development of several innovative vaccines against viral infections has been made possible by successful CRISPR/Cas9 editing of viral genomes. However, this gene-editing method has some side effects, one of which is off-target mutations. This manuscript summarises the ability of CRISPR/Cas9 to prevent and treat viral diseases in birds using recombinant vaccines and antiviral approaches. [ABSTRACT FROM AUTHOR]

Disruptions in normal development and the emergence of health conditions often result from the malfunction of vital genes in the human body. Decades of scientific research have focused on techniques to modify or substitute defective genes with healthy alternatives, marking a new era in disease treatment, prevention and cure. Recent strides in science and technology have reshaped our understanding of disorders, medication development and treatment recommendations, with human gene and cell therapy at the forefront of this transformative shift. Its primary objective is the modification of genes or adjustment of cell behaviour for therapeutic purposes. In this review, we focus on the latest advances in gene and cell therapy for treating human genetic diseases, with a particular emphasis on FDA and EMA‐approved therapies and the evolving landscape of genome editing. We examine the current state of innovative gene editing technologies, particularly the CRISPR‐Cas systems. As we explore the progress, ethical considerations and prospects of these innovations, we gain insight into their potential to revolutionize the treatment of genetic diseases, along with a discussion of the challenges associated with their regulatory pathways. This review traces the origins and evolution of these therapies, from conceptual ideas to practical clinical applications, marking a significant milestone in the field of medical science. [ABSTRACT FROM AUTHOR]

The rise in biological therapies has revolutionized oncology, with immunotherapy leading the charge through breakthroughs such as CAR-T cell therapy for melanoma and B-ALL. Modified bispecific antibodies and CAR-T cells are being developed to enhance their effectiveness further. However, CAR-T cell therapy currently relies on a costly ex vivo manufacturing process, necessitating alternative strategies to overcome this bottleneck. Targeted in vivo viral transduction offers a promising avenue but remains under-optimized. Additionally, novel approaches are emerging, such as in vivo vaccine boosting of CAR-T cells to strengthen the immune response against tumors, and dendritic cell-based vaccines are under investigation. Beyond CAR-T cells, mRNA therapeutics represent another promising avenue. Targeted delivery of DNA/RNA using lipid nanoparticles (LNPs) shows potential, as LNPs can be directed to T cells. Moreover, CRISPR editing has demonstrated the ability to precisely edit the genome, enhancing the effector function and persistence of synthetic T cells. Enveloped delivery vehicles packaging Cas9 directed to modified T cells offer a virus-free method for safe and effective molecule release. While this platform still relies on ex vivo transduction, using cells from healthy donors or induced pluripotent stem cells can reduce costs, simplify manufacturing, and expand treatment to patients with low-quality T cells. The use of allogeneic CAR-T cells in cancer has gained attraction for its potential to lower costs and broaden accessibility. This review emphasizes critical strategies for improving the selectivity and efficacy of immunotherapies, paving the way for a more targeted and successful fight against cancer. [ABSTRACT FROM AUTHOR]

BACKGROUND: The melon fly, Zeugodacus cucurbitae (Coquillett), is an invasive Tephritidae pest with robust fertility. The male accessory glands (MAGs) form a vital organ that ensures insect reproductive efficiency. Most of the secreted proteins by MAGs exhibit a male bias expression. Takeout, one of these proteins, is abundantly present in the MAGs of many insects. RESULTS: In this study, we identified 32 takeout genes in Z. cucurbitae. The phylogenetic analysis and multiple sequence alignment results showed that Zctakeout1 is the most related homolog to the MAGs‐specific takeout in Tephritidae. The real‐time quantitative PCR results showed that Zctakeout1 was exclusively expressed in the male adult stage, and its expression level gradually increased with the increase in age and then remained stable at the sexually matured stage. The distribution among tissues demonstrated the specific expression of Zctakeout1 in the MAGs, and fluorescence immunohistochemical results confirmed the presence of Zctakeout1 in close proximity to binuclear cells of the mesoderm epidermal MAGs. In continuation, CRISPR/Cas9‐mediated genome editing was employed, resulting in successfully generating a homozygous strain with an +8 bp insertion. The mating experiments with the Zctakeout1−/− males resulted in significant reductions in both the mating rate and egg production of females. CONCLUSION: These findings prove that the MAGs‐specific Zctakeout1 is essential in regulating fecundity in female Z. cucurbitae fruit flies. Our data suggests its utilization in future essential insect‐specific gene‐directed sterility insect technique (SIT) by the genetic manipulation to keep these important Tephritidae populations under control. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Saliva has a crucial role in determining the compatibility between piercing–sucking insects and their hosts. The brown planthopper (BPH) Nilaparvata lugens, a notorious pest of rice in East and Southeast Asia, secretes gelling and watery saliva when feeding on rice sap. Nlsalivap‐5 (NlSP5) and Nlsalivap‐7 (NlSP7) were identified as potential planthopper‐specific gelling saliva components, but their biological functions remain unknown. RESULTS: Here, we showed by transcriptomic analyses that NlSP5 and NlSP7 were biasedly expressed in the salivary glands of BPHs. Using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated genome‐editing system, we constructed NlSP5 and NlSP7 homozygous mutants (NlSP5−/− and NlSP7−/−). Electrical penetration graph assay showed that NlSP5−/− and NlSP7−/− mutants exhibited abnormal probing and feeding behaviors. Bioassays revealed that the loss‐of‐function of NlSP5 and NlSP7 significantly reduced the fitness of BPHs, with extended developmental duration, shortened lifespan, reduced weight, and impaired fecundity and hatching rates. CONCLUSION: These findings deepen our understanding of the BPH–host interaction and may provide potential targets for the management of rice planthoppers. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Climate change has rapidly increased incidences of frequent extreme abiotic stresses, such as heat, drought, salinity, and waterlogging. Each of these stressors negatively affects the cotton crop (Gossypium spp.) and results in significant yield decreases. Every stressful event causes specific changes in the metabolism and physiology of plants, which are linked to complex molecular alterations. Understanding the molecular mechanisms that regulate a plant's response to stress is essential to developing stress-resistant cotton varieties that can withstand various stress factors. Gene expressions in response to multiple stresses have been studied and mapped. These genes include ion transporters and heat shock proteins, which are vital to allowing adaptive responses. These approaches showed the ability to employ advanced genome sequencing and multi-omics techniques to identify dynamic gene expression patterns and elucidate intricate regulatory networks. Using genetic variation in combination with molecular techniques, it would be possible to generate stress-resilient cotton varieties that would enable sustainable cotton output in the face of abiotic stresses. Here, we reviewed the effects of major abiotic stressors on cotton plants, such as heat, salinity, drought, heavy metals, and waterlogging. We also examine the vast network of proteins, genes, and stress-sensitive signaling pathways that help cotton tolerate abiotic stress. [ABSTRACT FROM AUTHOR]

Summary The CYCS gene is highly evolutionarily conserved, with only a few pathogenic variants that cause thrombocytopenia‐4 (THC4). Here, we report a novel CYCS variant NM_018947.6: c.59C>T [NP_061820.1:p.(Thr20Ile)] segregating with thrombocytopenia in three generations of a Czech family. The phenotype of the patients corresponds to THC4 with platelets of normal size and morphology and dominant inheritance. Intriguingly, a gradual decline in platelet counts was observed across generations. CRISPR/Cas9‐mediated gene editing was used to introduce the new CYCS gene variant into a megakaryoblast cell line (MEG‐01). Subsequently, the adhesion, shape, size, ploidy, viability, mitochondrial respiration, cytochrome c protein (CYCS) expression, cell surface antigen expression and caspase activity were analysed in cells carrying the studied variant. Interestingly, the variant decreases the expression of CYCS while increasing mitochondrial respiration and the expression of CD9 cell surface antigen. Surprisingly, the variant abates caspase activation, contrasting with previously known effects of other CYCS variants. Some reports indicate that caspases may be involved in thrombopoiesis; thus, the observed dysregulation of caspase activity might contribute to thrombocytopenia. The findings significantly enhance our understanding of the molecular mechanisms underlying inherited thrombocytopenia and may have implications for diagnosis, prognosis and future targeted therapies. [ABSTRACT FROM AUTHOR]

The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus–host interactions and potential antiviral strategies. Importance: FMDV is a pathogen that causes highly contagious animal disease worldwide. The WD40 domain of ATG16L1 is necessary for non-canonical autophagy, while dispensable for canonical autophagy. Thus, the WD40 knockout PK15 cell line established by present study could use as a tool to research the impact of non-canonical autophagy on FMDV replication. The results showed that knockout of the WD40 domain of ATG16L1 in PK15 cells could enhance FMDV replication. The results also showed that differentially expressed innate immune factors, such as Mx1, IFIT1, RSAD2, and IRF9, were significantly enriched in the interferon signaling pathway. Our research also indicates non-canonical autophagy inhibits FMDV replication by innate immune, which helps to elucidate the role of the non-canonical autophagy in the host antiviral strategies. [ABSTRACT FROM AUTHOR]

SUMMARY: Chlamydomonas reinhardtii, a unicellular green alga, has been widely used as a model organism for studies of algal, plant and ciliary biology. The generation of targeted amino acid mutations is often necessary, and this can be achieved using CRISPR/Cas9 induced homology‐directed repair to install genomic modifications from exogenous donor DNA. Due to the low gene editing efficiency, the technical challenge lies in identifying the mutant cells. Direct sequencing is not practical, and pre‐screening is required. Here, we report a strategy for generating and screening for amino acid point mutations using the CRISPR/Cas9 gene editing system. The strategy is based on designing donor DNA using codon degeneracy, which enables the design of specific primers to facilitate mutant screening by PCR. An in vitro assembled RNP complex, along with a dsDNA donor and an antibiotic resistance marker, was electroporated into wild‐type cells, followed by PCR screening. To demonstrate this principle, we have generated the E102K mutation in centrin and the K40R mutation in α‐tubulin. The editing efficiencies at the target sites for Centrin, TUA1, TUA2 were 4, 24 and 8% respectively, based on PCR screening. More than 80% of the mutants with the expected size of PCR products were precisely edited, as revealed by DNA sequencing. Subsequently, the precision‐edited mutants were biochemically verified. The introduction of codon degeneracy did not affect the gene expression of centrin and α‐tubulins. Thus, this approach can be used to facilitate the identification of point mutations, especially in genes with low editing rates. [ABSTRACT FROM AUTHOR]

Simple Summary: Dmrt3 plays pivotal roles in testicular development, but the precise molecular mechanisms remain unclear. In this study, we investigated the role of medaka (Oryzias latipes) dmrt3 (dmrt3a) in testis development. This paper demonstrates that dmrt3a can maintain the number of germ cells and sperm motility, and it is expected to provide a theoretical basis for the diagnosis and treatment of human oligospermia and asthenospermia. Research across various species has demonstrated that the doublesex and mab-3-related transcription factor 3 (dmrt3) plays pivotal roles in testis development. However, the precise molecular mechanisms of dmrt3 remain unclear. In this study, we investigated the role of dmrt3 (dmrt3a) in testis development using the model organism medaka (Oryzias latipes). SqRT-PCR and ISH analyses revealed that dmrt3a is predominantly expressed in the testis, especially in the spermatid and spermatozoon. Using CRISPR/Cas9, we generated two dmrt3a homozygous mutants (-8 bp and -11 bp), which exhibited significantly reduced fertilization rates and embryo production. Additionally, the number of germ cells and sperm motility were markedly decreased in the dmrt3a mutants, manifesting as the symptoms of asthenozoospermia and oligozoospermia. Interestingly, RNA-Seq analysis showed that the deficiency of dmrt3a could lead to a significant downregulation of numerous genes related to gonadal development and severe disruptions in mitochondrial function. These results suggested that dmrt3a is essential for spermatogenesis and spermatozoa energy production. This paper provides new insights and perspectives for further exploring the molecular mechanisms underlying spermatogenesis and addressing male reproductive issues. [ABSTRACT FROM AUTHOR]

Environmental variations initiate chromatin modifications, leading to the exchange of histone subunits or the repositioning of nucleosomes. The phosphorylated histone variant H2A.X (γH2A.X) is recognized for the formation of foci that serve as established markers of DNA double-strand breaks (DSBs). Nevertheless, the precise roles of H2A.X in the cellular response to genotoxic stress and the impact of the plant hormone abscisic acid (ABA) remain incompletely understood. In this investigation, we implemented CRISPR/Cas9 technology to produce loss-of-function mutants of AtHTA3 and AtHTA5 in Arabidopsis. The phenotypes of the athta3 and athta5 single mutants were nearly identical to those of the wild-type Col-0. Nevertheless, the athta3 athta5 double mutants exhibited aberrant embryonic development, increased sensitivity to DNA damage, and higher sensitivity to ABA. The RT-qPCR analysis indicates that AtHTA3 and AtHTA5 negatively regulate the expression of AtABI3, a fundamental regulator in the ABA signaling pathway. Subsequent investigation demonstrated that AtABI3 participates in the genotoxic stress response by influencing the expression of DNA damage response genes, such as AtBRCA1, AtRAD51, and AtWEE1. Our research offers new insights into the role of H2A.X in the genotoxic and ABA responses of Arabidopsis. [ABSTRACT FROM AUTHOR]

Stargardt disease, one of the most common forms of inherited retinal diseases, affects individuals worldwide. The primary cause is mutations in the ABCA4 gene, leading to the accumulation of toxic byproducts in the retinal pigment epithelium (RPE) and subsequent photoreceptor cell degeneration. Over the past few years, research on Stargardt disease has advanced significantly, focusing on clinical and molecular genetics. Recent studies have explored various innovative therapeutic approaches, including gene therapy, stem cell therapy, and pharmacological interventions. Gene therapy has shown promise, particularly with adeno-associated viral (AAV) vectors capable of delivering the ABCA4 gene to retinal cells. However, challenges remain due to the gene's large size. Stem cell therapy aims to replace degenerated RPE and photoreceptor cells, with several clinical trials demonstrating safety and preliminary efficacy. Pharmacological approaches focus on reducing toxic byproduct accumulation and modulating the visual cycle. Precision medicine, targeting specific genetic mutations and pathways, is becoming increasingly important. Novel techniques such as clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 offer potential for directly correcting genetic defects. This review aims to synthesize recent advancements in understanding and treating Stargardt disease. By highlighting breakthroughs in genetic therapies, stem cell treatments, and novel pharmacological strategies, it provides a comprehensive overview of emerging therapeutic options. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 technology is expected to offer novel genome editing-related therapies for various diseases. We previously showed that an adenovirus vector (AdV) possessing eight expression units of multiplex guide RNAs (gRNAs) was obtained with no deletion of these units. Here, we attempted to construct "all-in-one" AdVs possessing expression units of four and eight gRNAs with Cas9 nickase, although we expected obstacles to obtain complete all-in-one AdVs. The first expected obstacle was that extremely high copies of viral genomes during replication may cause severe off-target cleavages of host cells and induce homologous recombination. However, surprisingly, four units in the all-in-one AdV genome were maintained completely intact. Second, for the all-in-one AdV containing eight gRNA units, we enlarged the E3 deletion in the vector backbone and shortened the U6 promoter of the gRNA expression units to shorten the AdV genome within the adenovirus packaging limits. The final size of the all-in-one AdV genome containing eight gRNA units still slightly exceeded the reported upper limit. Nevertheless, approximately one-third of the eight units remained intact, even upon preparation for in vivo experiments. Third, the genome editing efficiency unexpectedly decreased upon enlarging the E3 deletion. Our results suggested that complete all-in-one AdVs containing four gRNA units could be obtained if the problem of the low genome editing efficiency is solved, and those containing even eight gRNA units could be obtained if the obstacle of the vector size is also removed. [ABSTRACT FROM AUTHOR]

Hypertrophic cardiomyopathy (HCM) is a cardiovascular pathology that is caused by variants in genes encoding sarcomere-associated proteins. However, the clinical significance of numerous variants in HCM-associated genes is still unknown. CRISPR/Cas9 is a tool of nucleotide sequence editing that allows for the unraveling of different biological tasks. In this study, introducing a mutation with CRISPR/Cas9 into induced pluripotent stem cells (iPSCs) of a healthy donor and the directed differentiation of the isogenic iPSC lines into cardiomyocytes were used to assess the pathogenicity of a variant of unknown significance, p.M659I (c.1977G > A) in MYH7, which was found previously in an HCM patient. Using two single-stranded donor oligonucleotides with and without the p.M659I (c.1977G > A) mutation, together with CRISPR/Cas9, an iPSC line heterozygous at the p.M659I (c.1977G > A) variant in MYH7 was generated. No CRISPR/Cas9 off-target activity was observed. The iPSC line with the introduced p.M659I (c.1977G > A) mutation in MYH7 retained its pluripotent state and normal karyotype. Compared to the isogenic control, cardiomyocytes derived from the iPSCs with the introduced p.M659I (c.1977G > A) mutation in MYH7 recapitulated known HCM features: enlarged size, elevated diastolic calcium level, changes in the expression of HCM-related genes, and disrupted energy metabolism. These findings indicate the pathogenicity of the variant. [ABSTRACT FROM AUTHOR]

In the avian species, genetic modification by cell nuclear transfer is infeasible due to its unique reproductive system. The in vitro primordial germ cell modification approach is difficult and cumbersome, although it is the main method of genetic modification in chickens. In the present study, the adenoviral CRISPR/Cas9 vector was directly microinjected into the dorsal aorta of chicken embryos to achieve in vivo genetic modification. The results demonstrated that keratin 75-like 4 (KRT75L4), a candidate gene crucial for feather development, was widely knocked out, and an 8bp deletion was the predominant mutation that occurred in multiple tissues in chimeras, particularly in the gonad (2.63–11.57%). As we expected, significant modification was detected in the sperm of G0 (0.16–4.85%), confirming the potential to generate homozygous chickens and establishing this vector as a simple and effective method for genetic modification in avian species. [ABSTRACT FROM AUTHOR]

Purpose: Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is defined as a congenital visceral myopathy with genetic mutations. However, the etiology and pathophysiology are not fully understood. We aimed to generate a gene leiomodin-1a (lmod1a) modification technique to establish a zebrafish model of MMIHS. Methods: We targeted lmod1a in zebrafish using CRISPR/Cas9. After confirming the genotype, we measured the expression levels of the target gene and protein associated with MMIHS. A gut transit assay and spatiotemporal mapping were conducted to analyze the intestinal function. Results: Genetic confirmation showed a 5-base-pair deletion in exon 1 of lmod1a, which caused a premature stop codon. We observed significant mRNA downregulation of lmod1a, myh11, myod1, and acta2 and the protein expression of Lmod1 and Acta2 in the mutant group. A functional analysis of the lmod1a mutant zebrafish showed that its intestinal peristalsis was fewer, slower, and shorter in comparison to the wild type. Conclusion: This study showed that targeted deletion of lmod1a in zebrafish resulted in depletion of MMIHS-related genes and proteins, resulting in intestinal hypoperistalsis. This model may have the potential to be utilized in future therapeutic approaches, such as drug discovery screening and gene repair therapy for MMIHS. [ABSTRACT FROM AUTHOR]

Introduction and Purpose: In last years gene modification technologies such as CRISPR/Cas9 has had a revolutionary impact on the treatment of inherited diseases. Technologies developed from bacterial defense mechanisms, has become a basic tools in scientific research and medical therapies. In our article we provided an overview of applications of gene modifications technologies, directly focusing on CRISPR/Cas9, in genetic disease treatment. State of Knowledge: New applications of CRISPR/Cas9 are still being explored. Treating inherited diseases such as cystic fibrosis, Duchenne muscular dystrophy, thalassemia, hemophilia, Huntington's disease, Crigler-Najjar syndrome, sickle cell anemia, Marfan syndrome, and phenylketonuria, is feasible with this novel technique. A comparative analysis with other gene editing methods highlights CRISPR/Cas9's efficacy, ease of use, and multiplexing capabilities. Summary: CRISPR/Cas9 is a groundbreaking technology with broad applications in genetic research and therapy. Its ease of use, cost-effectiveness, and ability to target multiple genes simultaneously position it as a preferred method. However, there are some challenges associated with precision issues and ethical considerations in human embryo gene editing. As CRISPR/Cas9 continues to evolve, responsible application and ethical considerations are important for maximizing its potential in treatment of genetic diseases. [ABSTRACT FROM AUTHOR]

Tomato (Solanum lycopersicon L.) is one of the most widely consumed and produced vegetable crops worldwide. It offers numerous health benefits due to its rich content of many therapeutic elements such as vitamins, carotenoids, and phenolic compounds. Biotic stressors such as bacteria, viruses, fungi, nematodes, and insects cause severe yield losses as well as decreasing fruit quality. Conventional breeding strategies have succeeded in developing resistant genotypes, but these approaches require significant time and effort. The advent of state-of-the-art genome editing technologies, particularly CRISPR/Cas9, provides a rapid and straightforward method for developing high-quality biotic stress-resistant tomato lines. The advantage of genome editing over other approaches is the ability to make precise, minute adjustments without leaving foreign DNA inside the transformed plant. The tomato genome has been precisely modified via CRISPR/Cas9 to induce resistance genes or knock out susceptibility genes, resulting in lines resistant to common bacterial, fungal, and viral diseases. This review provides the recent advances and application of CRISPR/Cas9 in developing tomato lines with resistance to biotic stress. [ABSTRACT FROM AUTHOR]

The heading date, a critical trait influencing the rice yield and quality, has always been a hot topic in breeding research. Appropriately delaying the flowering time of excellent northern rice varieties is of great significance for improving yields and enhancing regional adaptability during the process for introducing varieties from north to south. In this study, genes influencing the heading date were identified through genome-wide association studies (GWAS). Using KenDao 12 (K12), an excellent cultivar from northern China, as the material, the specific flowering activator, OsMADS50, was edited using the genome-editing method to regulate the heading date to adapt to the southern planting environment. The results indicated that the osmads50 mutant line of K12 flowered about a week later, with a slight increase in the yield and good adaptability in the southern region in China. Additionally, the expressions of key flowering regulatory genes, such as Hd1, Ghd7, Ehd1, Hd3a, and RFT1, were reduced in the mutant plants, corroborating the delayed flowering phenotype. Yield trait analysis revealed that the primary factor for improved yield was an increase in the number of effective tillers, although there is potential for further enhancements in the seed-setting rate and grain plumpness. Furthermore, there were significant increases in the length-to-width ratio of the rice grains, fat content, and seed transparency, all contributing to an overall improvement in the rice quality. In summary, this study successfully obtained a rice variety with a delayed growth period through OsMADS50 gene editing, effectively implementing the strategy for adapting northern rice varieties to southern climates. This achievement significantly supports efforts to enhance the rice yield and quality as well as to optimize production management practices. [ABSTRACT FROM AUTHOR]

One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research. [ABSTRACT FROM AUTHOR]

Powdery mildew (PM), triggered by Oidium neolycopersici, represents a significant threat and a major concern for the productivity of tomato plants (Solanum lycopersicum L.). The presence of susceptibility (S) genes in plants facilitates pathogen proliferation and their dysfunction can lead to a recessively inherited broad-spectrum and durable type of resistance. Past studies have demonstrated that disrupting the function of DND1 (Defense No Death 1) increases plant resilience against various pathogens, such as powdery mildew (PM), but this comes at the cost of negatively affecting the overall health and vigor of the plant. To investigate the possibility of minimizing the adverse effects of the dnd1 mutation while boosting disease resistance, a CRISPR-Cas9 construct with four single guide RNAs targeting three exons of SlDND1 (Solyc02g088560.4.1) was designed and introduced into the tomato variety Moneymaker (MM) through Agrobacterium tumefaciens-mediated transformation. Three T1 lines (named E1, E3 and E4) were crossed with MM and then selfed to produce TF2 families. All the TF2 plants in homozygous state dnd1/dnd1, showed reduced PM symptoms compared to the heterozygous (DND1/dnd1) and wild type (DND1/DND1) ones. Two full knock-out (KO) mutant events (E1 and E4) encoding truncated DND1 proteins, exhibited clear dwarfness and auto-necrosis phenotypes, while mutant event E3 harbouring deletions of 3 amino acids, showed normal growth in height with less auto-necrotic spots. Analysis of the 3D structures of both the reference and the mutant proteins revealed significant conformational alterations in the protein derived from E3, potentially impacting its function. A dnd1/dnd1 TF2 line (TV181848-9, E3) underwent whole-genome sequencing using Illumina technology, which confirmed the absence of off-target mutations in selected genomic areas. Additionally, no traces of the Cas9 gene were detected, indicating its elimination through segregation. Our findings confirm the role of DND1 as an S-gene in tomato because impairment of this gene leads to a notable reduction in susceptibility to O. neolycopersici. Moreover, we provide, for the first time, a dnd1 mutant allele (E3) that exhibits fitness advantages in comparison with previously reported dnd1 mutant alleles, indicating a possible way to breed with dnd1 mutants. [ABSTRACT FROM AUTHOR]

The greater wax moth, Galleria mellonella (Lepidoptera, Pyralidae), is a major bee pest that inflicts considerable harm on beehives, leading to economic losses. It also serves as a valuable resource insect and a model organism. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system plays a crucial role in improving economic insect breeding and developing efficient agricultural pest management systems in Lepidoptera. However, the CRISPR/Cas9 protocols have not been developed for G. mellonella. Here, the Gmebony knockout (KO) strain was established using the CRISPR/Cas9 genome editing system. We obtained Gmebony KO strain in the G4 generation, which took approximately 10 months. When compared with wild‐type, the head, notum, and the terminal abdominal surface of 1st to 4th instar larvae in the KO strain changed from yellow to brown, and these regions of the KO strain gradually transformed into a black color from the 5th instar larvae, and the body color of the adult moth in the KO strain changed to black. The developmental period of the early larval and the following larval instars extended. The embryonic hatchability of the Gmebony KO strain was significantly decreased. The pupal body weight of the Gmebony KO strain was not affected. The feasibility of the CRISPR/Cas9 methodology was validated by single‐target editing of Gmebony. Our findings provide the first evidence that the ebony gene can serve as a pigmentation reference gene for genetic modifications of G. mellonella. Meanwhile, it can be utilized in the development of genome editing control strategies and for gene function analyses in G. mellonella. [ABSTRACT FROM AUTHOR]

Monogalactosyldiacylglycerol (MGDG), a predominant photosynthetic membrane lipid derived from plants and microalgae, has important applications in feed additives, medicine, and other fields. The low content and various structural stereoselectivity differences of MGDG in plants limited the biological extraction or chemical synthesis of MGDG, resulting in a supply shortage of monogalactosyldiacylglycerol with a growing demand. Herein, we established Saccharomyces cerevisiae as a cell factory for efficient de novo production of monogalactosyldiacylglycerol for the first time. Heterologous production of monogalactosyldiacylglycerol was achieved by overexpression of codon-optimized monogalactosyldiacylglycerol synthase gene MGD1, the key Kennedy pathway genes (i.e. GAT1, ICT1, and PAH1), and multi-copy integration of the MGD1 expression cassette. The final engineered strain (MG-8) was capable of producing monogalactosyldiacylglycerol with titers as high as 16.58 nmol/mg DCW in a shake flask and 103.2 nmol/mg DCW in a 5 L fed-batch fermenter, respectively. This is the first report of heterologous biosynthesis of monogalactosyldiacylglycerol in microorganisms, which will provide a favorable reference for study on heterologous production of monogalactosyldiacylglycerol in yeasts. [ABSTRACT FROM AUTHOR]

Background: Photoperiod sensitivity is among the most important agronomic traits of rice, as it determines local and seasonal adaptability and plays pivotal roles in determining yield and other key agronomic characteristics. By controlling the photoperiod, early-maturing rice can be cultivated to shorten the breeding cycle, thereby reducing the risk of yield losses due to unpredictable climate change. Furthermore, early-maturing and high-yielding rice needs to be developed to ensure food security for a rapidly growing population. Early-maturing and high-yielding rice should be developed to fulfill these requirements. OsCKq1 encodes the casein kinase1 protein in rice. OsCKq1 is a gene that is activated by photophosphorylation when Ghd7, which suppresses flowering under long-day conditions, is activated. Results: This study investigates how OsCKq1 affects heading in rice. OsCKq1-GE rice was analyzed the function of OsCKq1 was investigated by comparing the expression levels of genes related to flowering regulation. The heading date of OsCKq1-GE lines was earlier (by about 3 to 5 days) than that of Ilmi (a rice cultivar, Oryza sativa spp. japonica), and the grain length, grain width, 1,000-grain weight, and yield increased compared to Ilmi. Furthermore, the culm and panicle lengths of OsCKq1-GE lines were either equal to or longer than those of Ilmi. Conclusions: Our research demonstrates that OsCKq1 plays a pivotal role in regulating rice yield and photoperiod sensitivity. Specifically, under long-day conditions, OsCKq1-GE rice exhibited reduced OsCKq1 mRNA levels alongside increased mRNA levels of Hd3a, Ehd1, and RFT1, genes known for promoting flowering, leading to earlier heading compared to Ilmi. Moreover, we observed an increase in seed size. These findings underscore OsCKq1 as a promising target for developing early-maturing and high-yielding rice cultivars, highlighting the potential of CRISPR/Cas9 technology in enhancing crop traits. [ABSTRACT FROM AUTHOR]

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system, a groundbreaking innovation in genetic engineering, has revolutionized our approach to surmounting complex diseases, culminating in CASGEVY™ approved for sickle cell anemia. Derived from a microbial immune defense mechanism, CRISPR/Cas9, characterized as precision, maneuverability and universality in gene editing, has been harnessed as a versatile tool for precisely manipulating DNA in mammals. In the process of applying it to practice, the consecutive exploitation of novel orthologs and variants never ceases. It's conducive to understanding the essentialities of diseases, particularly cancer, which is crucial for diagnosis, prevention, and treatment. CRISPR/Cas9 is used not only to investigate tumorous genes functioning but also to model disparate cancers, providing valuable insights into tumor biology, resistance, and immune evasion. Upon cancer therapy, CRISPR/Cas9 is instrumental in developing individual and precise cancer therapies that can selectively activate or deactivate genes within tumor cells, aiming to cripple tumor growth and invasion and sensitize cancer cells to treatments. Furthermore, it facilitates the development of innovative treatments, enhancing the targeting efficiency of reprogrammed immune cells, exemplified by advancements in CAR-T regimen. Beyond therapy, it is a potent tool for screening susceptible genes, offering the possibility of intervening before the tumor initiative or progresses. However, despite its vast potential, the application of CRISPR/Cas9 in cancer research and therapy is accompanied by significant efficacy, efficiency, technical, and safety considerations. Escalating technology innovations are warranted to address these issues. The CRISPR/Cas9 system is revolutionizing cancer research and treatment, opening up new avenues for advancements in our understanding and management of cancers. The integration of this evolving technology into clinical practice promises a new era of precision oncology, with targeted, personalized, and potentially curative therapies for cancer patients. [ABSTRACT FROM AUTHOR]

The leaf is a determinate organ with a final size under genetic control. Numerous factors that regulate the final leaf size have been identified in Arabidopsis thaliana ; although most of these factors play their roles during the growth of leaf primordia, much less is known about leaf initiation and its effects on the final leaf size. In this study, we characterized oligocellula6-D (oli6-D), a semidominant mutant of A. thaliana with smaller leaves than the wild type (WT) due to its reduced leaf cell numbers. A time-course analysis showed that oli6-D had approximately 50% fewer leaf cells even immediately after leaf initiation; this difference was maintained throughout leaf development. Next-generation sequencing showed that oli6-D had chromosomal duplications involving 2-kb and 3-Mb regions of chromosomes 2 and 4, respectively. Several duplicated genes examined had approximately 2-fold higher expression levels, and at least one gene acquired a new intron/exon structure due to a chromosome fusion event. oli6-D showed reduced auxin responses in leaf primordia, primary roots and embryos, as well as reduced apical dominance and partial auxin-resistant root growth. CRISPR-associated protein-9-mediated genome editing enabled the removal of a 3-Mb duplicated segment, the largest targeted deletion in plants thus far. As a result, oli6-D restored the WT leaf phenotypes, demonstrating that oli6-D is a gain-of-function mutant. Our results suggest a new regulatory point of leaf size determination that functions at a very early stage of leaf development and is negatively regulated by one or more genes located in the duplicated chromosomal segments. [ABSTRACT FROM AUTHOR]

GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of β‐galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH‐SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock‐out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP‐CHOP websites, and high‐efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH‐SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X‐gal and Periodic acid‐Schiff (PAS) staining, and qPCR were used to confirm the knock‐out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock‐out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH‐SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non‐transfected cell, X‐gal staining resulted in no blue color in GLB1 gene knock‐out cells indicating the absence of β‐galactosidase enzyme activity in these cells. Real‐time PCR (qPCR) results confirmed the RNA‐Seq analysis outcomes on the GEO data set and following the GLB1 gene knock‐out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1‐NEU1‐CTSA complex gene was involved in suppressed proliferation and invasion ability of knock‐out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH‐SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease. Significance statement: This study concerns the modeling of GM1 gangliosidosis by knocking‐out the GLB1 gene in human nerve cells and using CRISPR/Cas9 technology. [ABSTRACT FROM AUTHOR]

MicroRNAs play critical roles in multiple developmental processes in insects. Our previous study showed that CRISPR/Cas9‐mediated knock down of the microRNA let‐7 in silkworms increased the size of larvae and silk glands, thereby improving the silk production capacity. In this study, we elucidate the molecular mechanism underlying of let‐7 regulates growth. Identification of differentially expressed genes in response to let‐7 knock down revealed enrichment of pathways associated with cell proliferation and DNA replication. let‐7 dysregulation affected the cell cycle and proliferation of the Bombyx mori cell line BmN. Dual‐luciferase and target site mutation assays showed that BmCDK1 is a direct target gene of let‐7, with only 1 binding site on its 3′‐untranslated region. RNA interference of BmCDK1 inhibited cell proliferation, but this effect was counteracted by co‐transfection with let‐7 antagomir. Moreover, let‐7 knock down induced BmCDK1 expression and promoted cell proliferation in multiple tissues, and further induced endomitosis in the silk gland in vivo. Knock down of BmCDK1 resulted in abnormal formation of a new epidermis, and larval development was arrested at the 2nd or 3rd molt stage. Taken together, our results demonstrated that BmCDK1 is a novel target of let‐7 in cell fate determination, possessing potential for improving silk yield in silkworm. [ABSTRACT FROM AUTHOR]

Although CRISPR/Cas9 has been widely used in insect gene editing, the need for the microinjection of preblastoderm embryos can preclude the technique being used in insect species with eggs that are small, have hard shells, and/or are difficult to collect and maintain outside of their normal environment. Such is the case with Sogatella furcifera, the white‐backed planthopper (WBPH), a significant pest of Oryza sativa (rice) that oviposits inside rice stems. Egg extraction from the stem runs the risk of mechanical damage and hatching is heavily influenced by the micro‐environment of the rice stem. To bypass these issues, we targeted embryos prior to oviposition via direct parental (DIPA)‐CRISPR, in which Cas9 and single‐guide RNAs (sgRNAs) for the WBPH eye pigment gene tryptophan 2,3‐dioxygenase were injected into the hemocoel of adult females. Females at varying numbers of days posteclosion were evaluated to determine at what stage their oocyte might be most capable of taking up the gene‐editing components. An evaluation of the offspring indicated that the highest G0 gene‐edited efficacy (56.7%) occurred in females injected 2 d posteclosion, and that those mutations were heritably transmitted to the G1 generation. This study demonstrates the potential utility of DIPA‐CRISPR for future gene‐editing studies in non‐model insect species and can facilitate the development of novel pest management applications. [ABSTRACT FROM AUTHOR]

Key message: The Osckx2 mutant accumulates cytokinin thereby enhancing panicle branching, grain yield, and drought tolerance, marked by improved survival rate, membrane integrity, and photosynthetic function. Cytokinins (CKs) are multifaceted hormones that regulate growth, development, and stress responses in plants. Cytokinins have been implicated in improved panicle architecture and grain yield; however, they are inactivated by the enzyme cytokinin oxidase (CKX). In this study, we developed a cytokinin oxidase 2 (Osckx2)-deficient mutant using CRISPR/Cas9 gene editing in indica rice and assessed its function under water-deficit and salinity conditions. Loss of OsCKX2 function increased grain number, secondary panicle branching, and overall grain yield through improved cytokinin content in the panicle tissue. Under drought conditions, the Osckx2 mutant conserved more water and demonstrated improved water-saving traits. Through reduced transpiration, Osckx2 mutants showed an improved survival response than the wild type to unset dehydration stress. Further, Osckx2 maintained chloroplast and membrane integrity and showed significantly improved photosynthetic function under drought conditions through enhanced antioxidant protection systems. The OsCKX2 function negatively affects panicle grain number and drought tolerance, with no discernible impact in response to salinity. The finding suggests the utility of the beneficial Osckx2 allele in breeding to develop climate-resilient, high-yielding cultivars for future food security. [ABSTRACT FROM AUTHOR]

Derivation of hypoimmunogenic human cells from genetically manipulated pluripotent stem cells holds great promise for future transplantation medicine and adoptive immunotherapy. Disruption of beta-2-microglobulin (B2M) in pluripotent stem cells followed by differentiation into specialized cell types is a promising approach to derive hypoimmunogenic cells. Given the attractive features of CRISPR/Cas9-based gene editing tool and baculoviral delivery system, baculovirus can deliver CRISPR/Cas9 components for site-specific gene editing of B2M. Herein, we report the development of a baculoviral CRISPR/Cas9 vector system for the B2M locus disruption in human cells. When tested in human embryonic stem cells (hESCs), the B2M gene knockdown/out was successfully achieved, leading to the stable down-regulation of human leukocyte antigen class I expression on the cell surface. Fibroblasts derived from the B2M gene-disrupted hESCs were then used as stimulator cells in the co-cultures with human peripheral blood mononuclear cells. These fibroblasts triggered significantly reduced alloimmune responses as assessed by sensitive Elispot assays. The B2M-negative hESCs maintained the pluripotency and the ability to differentiate into three germ lineages in vitro and in vivo. These findings demonstrated the feasibility of using the baculoviral-CRISPR/Cas9 system to establish B2M-disrupted pluripotent stem cells. B2M knockdown/out sufficiently leads to hypoimmunogenic conditions, thereby supporting the potential use of B2M-negative cells as universal donor cells for allogeneic cell therapy. [ABSTRACT FROM AUTHOR]

Summary: Eukaryotic translation initiation factors (eIFs) are important for mRNA translation but also pivotal for plant‐virus interaction. Most of these plant‐virus interactions were found between plant eIFs and the viral protein genome‐linked (VPg) of potyviruses. In case of lost interaction due to mutation or deletion of eIFs, the viral translation and subsequent replication within its host is negatively affected, resulting in a recessive resistance. Here we report the identification of the Beta vulgaris Bv‐eIF(iso)4E as a susceptibility factor towards the VPg‐carrying beet chlorosis virus (genus Polerovirus). Using yeast two‐hybrid and bimolecular fluorescence complementation assays, the physical interaction between Bv‐eIF(iso)4E and the putative BChV‐VPg was detected, while the VPg of the closely related beet mild yellowing virus (BMYV) was found to interact with the two isoforms Bv‐eIF4E and Bv‐eIF(iso)4E. These VPg‐eIF interactions within the polerovirus‐beet pathosystem were demonstrated to be highly specific, as single mutations within the predicted cap‐binding pocket of Bv‐eIF(iso)4E resulted in a loss of interaction. To investigate the suitability of eIFs as a resistance resource against beet infecting poleroviruses, B. vulgaris plants were genome edited by CRISPR/Cas9 resulting in knockouts of different eIFs. A simultaneous knockout of the identified BMYV‐interaction partners Bv‐eIF4E and Bv‐eIF(iso)4E was not achieved, but Bv‐eIF(iso)4EKO plants showed a significantly lowered BChV accumulation and decrease in infection rate from 100% to 28.86%, while no influence on BMYV accumulation was observed. Still, these observations support that eIFs are promising candidate genes for polerovirus resistance breeding in sugar beet. [ABSTRACT FROM AUTHOR]