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In utero gene editing with mRNA-based therapeutics has the potential to revolutionize the treatment of neurodevelopmental disorders. However, a critical bottleneck in clinical application has been the lack of mRNA delivery vehicles that can efficiently transfect cells in the brain. In this report, we demonstrate that in utero intracerebroventricular (ICV) injection of densely PEGylated lipid nanoparticles (ADP-LNPs) containing an acid-degradable PEG-lipid can safely and effectively deliver mRNA for gene editing enzymes to the fetal mouse brain, resulting in successful transfection and editing of brain cells. ADP-LNPs containing Cre mRNA transfected 30% of the fetal brain cells in Ai9 mice and had no detectable adverse effects on fetal development and postnatal growth. In addition, ADP-LNPs efficiently transfected neural stem and progenitor cells in Ai9 mice with Cre mRNA, which subsequently proliferated and caused over 40% of the cortical neurons and 60% of the hippocampal neurons to be edited in treated mice 10 weeks after birth. Furthermore, using Angelman syndrome, a paradigmatic neurodevelopmental disorder, as a disease model, we demonstrate that ADP-LNPs carrying Cas9 mRNA and gRNA induced indels in 21% of brain cells within 7 days postpartum, underscoring the precision and potential of this approach. These findings demonstrate that LNP/mRNA complexes have the potential to be a transformative tool for in utero treatment of neurodevelopmental disorders and set the stage for a frontier in treating neurodevelopmental disorders that focuses on curing genetic diseases before birth.

Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.

Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker-free genome edited Nicotiana benthamiana N-glycosylation mutant (NbXF-KO) carrying inactivated β1,2-xylosyltransferase and α1,3-fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing. Mass spectrometric analyses showed the synthesis of N-glycans devoid of plant-specific β1,2-xylose and core α 1,3-fucose on endogenous proteins and a series of recombinantly expressed glycoproteins with different complexities. Further transient glycan engineering towards more diverse human-type N-glycans resulted in the production of recombinant proteins decorated with β1,4-galactosylated and α2,6-sialylated structures, respectively. Notably, a monoclonal antibody expressed in the NbXF-KO displayed glycosylation-dependent activities. Collectively, the engineered plants grow normally and are well suited for upscaling, thereby meeting industrial and regulatory requirements for the production of high-quality therapeutic proteins.

Epigenetics refers to the molecules and mechanisms that modify gene expression states without changing the nucleotide context. These modifications are what encode the cell state during differentiation or epigenetic memory in mitosis. Epigenetic modifications can alter gene expression by changing the chromatin architecture by altering the affinity for DNA to wrap around histone octamers, forming nucleosomes. The higher affinity the DNA has for the histones, the tighter it will wrap and therefore induce a heterochromatin state, silencing gene expression. Several groups have shown the ability to harness the cell's natural epigenetic modification pathways to engineer proteins that can induce changes in epigenetics and consequently regulate gene expression. Therefore, epigenetic modification can be used to target and treat disorders through the modification of endogenous gene expression. The use of epigenetic modifications may prove an effective path towards regulating gene expression to potentially correct or cure genetic disorders.

Cryomyces antarcticus, a melanized cryptoendolithic fungus endemic to Antarctica, can tolerate environmental conditions as severe as those in space. Particularly, its ability to withstand ionizing radiation has been attributed to the presence of thick and highly melanized cell walls, which—according to a previous investigation—may contain both 1,8‐dihydroxynaphthalene (DHN) and L‐3,4 dihydroxyphenylalanine (L‐DOPA) melanin. The genes putatively involved in the synthesis of DHN melanin were identified in the genome of C. antarcticus. Most important is capks1 encoding a non‐reducing polyketide synthase (PKS) and being the ortholog of the functionally characterized kppks1 from the rock‐inhabiting fungus Knufia petricola. The co‐expression of CaPKS1 or KpPKS1 with a 4′‐phosphopantetheinyl transferase in Saccharomyces cerevisiae resulted in the formation of a yellowish pigment, suggesting that CaPKS1 is the enzyme providing the precursor for DHN melanin. To dissect the composition and function of the melanin layer in the outer cell wall of C. antarcticus, non‐melanized mutants were generated by CRISPR/Cas9‐mediated genome editing. Notwithstanding its slow growth (up to months), three independent non‐melanized Δcapks1 mutants were obtained. The mutants exhibited growth similar to the wild type and a light pinkish pigmentation, which is presumably due to carotenoids. Interestingly, visible light had an adverse effect on growth of both melanized wild‐type and non‐melanized Δcapks1 strains. Further evidence that light can pass the melanized cell walls derives from a mutant expressing a H2B‐GFP fusion protein, which can be detected by fluorescence microscopy. In conclusion, the study reports on the first genetic manipulation of C. antarcticus, resulting in non‐melanized mutants and demonstrating that the melanin is rather of the DHN type. These mutants will allow to elucidate the relevance of melanization for surviving extreme conditions found in the natural habitat as well as in space. [ABSTRACT FROM AUTHOR]

The article discusses the development of a gene editing system in the economic macroalga Neopyropia yezoensis using CRISPR/Cas9 technology. The study aims to improve breeding strategies for N. yezoensis, which has high economic value and unique biological properties. The successful editing of a target gene in N. yezoensis using CRISPR/Cas9 lays the foundation for molecular breeding and positions the species as a potential model organism for marine algae studies. The study demonstrates the efficiency of the gene editing system and its potential impact on the seaweed industry. [Extracted from the article]

Animal silk is economically important, while silk secretion is a complex and subtle mechanism regulated by many genes. We identified the poly (ADP‐ribose) polymerase (PARP1) gene of the silkworm and successfully cloned its coding sequence (CDS) sequence. Using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) technology, we screened single guide RNA (sgRNA) with high knockout efficiency by cellular experiments and obtained PARP1 mutants by knocking out the PARP1 gene of the silkworm at the individual level. We found that the mutants mainly exhibited phenotypes such as smaller cocoon size and reduced cocoon shell rate than the wild type. We also detected the expression of silk protein genes in the mutant by quantitative real‐time PCR (qPCR) and found that the expression of some silk protein genes was slightly down‐regulated. Meanwhile, together with the results of transcriptomic analysis, we hypothesized that PARP1 may affect the synthesis of silk proteins, resulting in their failure to function properly. Our study may provide an important reference for future in‐depth refinement of the molecular mechanism of silk protein expression in silk‐producing animals, as well as a potential idea for future development of molecular breeding lines of silkworms to improve silk production. [ABSTRACT FROM AUTHOR]

Insect reproductive capacity can affect effective pest control and infertility studies and has become an important focus in recent molecular genetic research. Nucleosome assembly protein (Nap) is highly conserved across multiple species and is involved in forming the sperm nucleus in many species. We used clustered regularly interspaced palindromic repeats/Cas9 technology to knockout BmNap in Bombyx mori and observed that the mutations caused female infertility, whereas male fertility was not affected. BmNap mutants grew and mated normally; however, female mutants laid smaller eggs that could not be fertilised and did not hatch. In addition, female sterility produced by the mutation could be inherited stably via male mutants; therefore, Nap could be used as a potential target for lepidopteran pest control through population regulation. In the current study, we elucidated a new function of BmNap, increased the understanding of the oogenesis regulation network in Lepidoptera and promoted the development of insect sterility technologies. [ABSTRACT FROM AUTHOR]

Plants acts as living chemical factories that may create a large variety of secondary metabolites, most of which are used in pharmaceutical products. The production of these secondary metabolites is often much lower. Moreover, the primary constraint after discovering potential metabolites is the capacity to manufacture sufficiently for use in industrial and therapeutic contexts. The development of omics technology has brought revolutionary discoveries in various scientific fields, including transcriptomics, metabolomics, and genome sequencing. The metabolic pathways leading to the utilization of new secondary metabolites in the pharmaceutical industry can be identified with the use of these technologies. Genome editing (GEd) is a versatile technology primarily used for site-directed DNA insertions, deletions, replacements, base editing, and activation/repression at the targeted locus. Utilizing GEd techniques such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9), metabolic pathways engineered to synthesize bioactive metabolites optimally. This article will briefly discuss omics and CRISPR/Cas9-based methods to improve secondary metabolite production in medicinal plants. [ABSTRACT FROM AUTHOR]

Genome editing, mainly CRISPR/Cas9, has been revolutionizing agricultural biotechnology through precise modifications to plant and animal genomes. This review highlights advancements in research and regulatory development of genome editing across Latin America. The region has seen substantial progress in building a regulatory framework to adopt genome editing technologies that enhance crop yield, nutritional content, and resistance to pests and diseases, as well as address critical challenges such as food insecurity and climate change. The article discusses the evolution of regulatory frameworks in various Latin American countries and their trends toward using foreign genetic material to determine regulatory oversight. Argentina pioneered this approach, followed by Brazil, Colombia, Chile, Paraguay, Honduras, and Guatemala. Recent updates from Costa Rica, El Salvador, Ecuador, Uruguay, and Colombia reflect a similar rationale to balance innovation with safety and compliance with international standards. Regional examples of genome editing demonstrate the potential to improve crop quality and sustainability of coffee, rice, cacao, beans, and potatoes. Public perception and acceptance of genome editing are also explored, with surveys indicating strong support for regulatory measures in Costa Rica. The article further explores public perception and acceptance of genome editing indicating strong support for regulation in Costa Rica. The review underscores the importance of continued research, public engagement, and international cooperation that foster responsible development of genome-editing technologies in Latin America. Key message: Genome editing advances in Latin America showcase regulatory progress and public support, empowering agricultural innovation to boost crop resilience, yield, and sustainability, while addressing food security and climate challenges. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection remains a significant global health challenge, with chronic HBV leading to severe liver diseases, including cirrhosis and hepatocellular carcinoma. Current treatments often fail to eradicate the virus, highlighting the need for innovative therapeutic strategies. The CRISPR/Cas9 system has emerged as a dynamic tool for precise genome editing and presents a promising approach to targeting and eliminating HBV infection. This review provides a comprehensive overview of the advances, challenges, and delivery strategies associated with CRISPR/Cas9-based therapies for HBV. We begin by elucidating the mechanism of the CRISPR/Cas9 system and then explore HBV pathogenesis, focusing on the role of covalently closed circular DNA (cccDNA) and integrated HBV DNA in maintaining chronic infection. CRISPR/Cas9 can disrupt these key viral reservoirs, which are critical for persistent HBV replication and associated liver damage. The application of CRISPR/Cas9 in HBV treatment faces significant challenges, such as off-target effects, delivery efficiency, and immune responses. These challenges are addressed by examining current approaches to enhance the specificity, safety, and efficacy of CRISPR/Cas9. A future perspective on the development and clinical translation of CRISPR/Cas9 therapies for HBV is provided, emphasizing the requirement for further research to improve delivery methods and ensure durable safety and effectiveness. This review underscores the transformative potential of CRISPR/Cas9 in combating HBV and sets the stage for future breakthroughs in the field. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 system represents a state-of-the-art technology for precise genome editing in plants. In this study, we performed in silico and evolutionary analyses, as well as designed guide RNA (gRNA) constructs for the precise modification of the thermosensitive genic male sterile (OsTMS5) gene using the CRISPR/Cas9 system in rice (Oryza sativa L.). The OsTMS5 promoter harbours a diverse array of cis-elements, which are linked to light responsiveness, hormonal regulation, and stress-related signaling. Further, expression pattern of OsTMS5 revealed that OsTMS5 exhibited responsiveness to hormones and was activated across diverse tissues and developmental stages in rice. In addition, we meticulously designed gRNA with a length of 20 base pairs. This design process was conducted using the CRISPR-P v2.0 online platform. The target of these gRNAs was the rice OsTMS5 gene. The selection of the top two gRNAs was made after conducting a thorough evaluation, which included assessing factors such as on-score value, minimum off-target score, GC content, potential off-target sites, and genomic location. Furthermore, two types of entry vectors were utilized, and the pMDC99 vector served as the destination vector for plant transformation. Following the annealing and ligation of the gRNAs through LR recombination, the resulting plasmid was named as "pMDC99-eSPCas9 + OsU6-OsTMS5-target1-gRNA + OsU6-OsTMS5-target2-gRNA". Subsequently, this plasmid obtained from the third LR recombination was introduced into Agrobacterium EHA105 for the purpose of conducting rice transformation. Therefore, these constructs have the potential for use not only in molecular genetic analyses and molecular breeding in rice but also in a wide range of other crop species. [ABSTRACT FROM AUTHOR]

The environmental conditions play a crucial role in determining crop yield, which is essential for ensuring food and nutritional security. However, rapid climate change is exacerbating global environmental stress, leading to severe biotic pressures on crops. Therefore, enhancing crop resilience to pathogens has become one of the most pressing challenges for humanity. Large-scale mutant library screening is the most efficient strategy for identifying numerous genes associated with specific traits. The revolutionary CRISPR/Cas9 system has ushered in a new era in the construction of mutant library. However, its application in crop plants has been relatively scarce compared to mammals, largely due to challenges in accessibility. Fortunately, several research groups have recently developed CRISPR/Cas9-based mutant libraries, successfully identifying a variety of genes involved in crop immunity. In this review, we present an overview and discussion of studies that have generated significant results through the use of CRISPR/Cas9 library screening to identify novel genes associated with resistance to biotic stresses within the field of plant research. [ABSTRACT FROM AUTHOR]

Introduction: Tuberculosis (TB) is a serious infectious disease that endangers human health, and TB becomes more difficult in eradiation due to its multidrug resistance (MDR). The objective of this research was to identify novel targets for treating TB. Methods: A 2-fold serial dilution method was used to determine minimal inhibitory concentrations (MIC) of compound M6 against Mycobacterium smegmatis (M. smegmatis). Compound M6 was subjected to reverse molecular docking with seven Mycobacterium tuberculosis proteins, and the best binding protein with the highest LibDock score was evaluated. The target protein with the highest score was purified through prokaryotic expression. Isolated target proteins were investigated for the enzyme activities and for the kinetic effect of compound M6 by absorbance detection. Subsequently, the CRISPR/Cas9 technology was employed to inhibit target gene expression for detecting MIC changes. Finally, potential targets were evaluated for the effect of the compound M6 in bacteria. Results: The MIC values of compound M6 against M. smegmatis were 32 μg/mL. The results from reverse molecular docking show that IspD has the highest LibDock score of 142.50, followed by Rv0674, IspF, and Dxr, with docking scores of 110.762, 71.6955, and 57.7446, respectively. IspD is a key enzyme in the 2-C-methyl-D-erythritol 4-phosphate pathway of MTB. The aKi and Ki values of M6 for the substrate MEP are 609.58 μM and 81.33 μM. For CTP, the aKi and Ki values are 657.89 μM and 40.07 μM. With tetracycline inducing CRISPR/Cas9 to suppress the expression of IspD, the MIC value of M6 against IspD went down significantly from 32 μg/mL to 4 μg/mL. Conclusion: IspD is a novel target of the compound M6 for treating TB. [ABSTRACT FROM AUTHOR]

SUMMARY The glutamate receptor (GLR) serves as a ligand‐gated ion channel that plays a vital role in plant growth, development, and stress response. Nevertheless, research on GLRs in cotton is still very limited. The present study conducted a comprehensive analysis of GLRs gene family in cotton. In total, 41 members of the GLR family were identified in cotton unveiling distinct subgroups in comparison to Arabidopsis. Among these members, the third subgroup highlights its pivotal role in cotton's defense against insect infestation. Furthermore, the CRISPR/Cas9 system was utilized to create a mutant library of GLR members, which consisted of a total of 135 independent mutant lines, resulting in the production of novel cotton materials with valuable breeding potential for pest control. Further, this study elucidates the influence of GhGLR3.4 on jasmonic acid (JA) pathway signal transduction and demonstrated its participation in the influx of intracellular Ca2+, which regulates “calcium transients” following stimulation, thereby influencing multiple intracellular reactions. The study also found that GhGLR3.4 influences the synthesis of the JA pathway and actively partakes in long‐distance signal transmission among plants, facilitating the transfer of defense signals to neighbor leaves and thereby triggering systemic defense. Consequently, this research advances our knowledge of plants' comprehensive defense mechanism against insect pest infestation. [ABSTRACT FROM AUTHOR]

ABSTRACT Missing an entire chromosome or chromosome arm in normal diploid cells has a deleterious impact on cell viability, which may contribute to the development of specific birth defects. Nevertheless, the effects of chromosome loss in human cells have remained unexplored due to the lack of suitable model systems. Here, we developed an efficient, selection‐free approach to generate partial monosomy in human induced pluripotent stem cells (iPSCs). The introduction of Cas9 proteins and a pair of gRNAs induces over megabase‐sized interstitial chromosomal deletions. Using human chromosome 21 (HSA21) as a model, partial monosomy 21q (PM21q) iPSC lines with deletions ranging from 4.5 to 27.9 Mb were isolated. A 33.6 Mb deletion, encompassing all protein‐coding genes on 21q, was also achieved, establishing the first 21q monosomy human iPSC line. Transcriptome and proteome analyses revealed that the abundances of mRNA and protein encoded by the majority of genes in the monosomic regions are half of the diploid expression level, indicating an absence of dosage compensation. The ability to generate customized partial monosomy cell lines on an isogenic, karyotypically normal background should facilitate the gain of novel insights into the impact of chromosome loss on cellular fitness. [ABSTRACT FROM AUTHOR]

As a major component of tumor infiltration, tumor‐associated macrophages (TAMs) primarily promote the M2 phenotype of tumors in the tumor microenvironment. Additionally, the overexpression of anti‐phagocytic molecules in TAMs facilitates tumor cell evasion of TAMs' phagocytic arrest. Therefore, there is an urgent need to develop a reliable strategy to reprogram TAMs for more effective anti‐tumor outcomes. In this study, innovative inhalable bioorthogonal gene‐editable microparticles (SPR) have been engineered that reprogram TAMs for lung cancer therapy based on spike‐covered sunflower sporopollenin exine capsules (SECs). These microparticles utilize SECs as delivery vehicles, with reduced palladium (Pd) nanoparticles on their surface carrying CRISPR/Cas9 lipid nanoparticles (LNP‐RNPs) that specifically knock down the anti‐phagocytic SIRPα gene on TAMs. The SPR microparticles employ a tripartite strategy to reprogram the TAMs: the physical stimulation through pollen spikes, the production of a Toll‐like receptor 7 agonist (imiquimod, IMD) via Pd‐mediated bioorthogonal catalysis, and the employment of CRISPR/Cas9 gene editing. In an orthotopic mouse model, the inhalation of SPR microparticles not only reprograms macrophages efficiently but also bolsters their tumor‐fighting abilities. Notably, the cured mice show no signs of recurrence even upon re‐exposure to the tumor, indicating a long‐lasting anti‐tumor effect. [ABSTRACT FROM AUTHOR]

Increasing population and climate change pose significant threats to global food security by imposing stresses on plants, making them more susceptible to diseases and productivity losses caused by pathogens, pests, and weeds. Traditional breeding strategies are insufficient for rapid development of new plant traits that can outpace this productivity downtrend. Modern advances in genome editing technologies, particularly CRISPR/Cas9, have revolutionised crop protection through precise and targeted genome modifications. This allows for the development of resilient crops with enhanced resistance against pathogens, pests, and weeds. This review explores various approaches with which CRISPR/Cas9 is applied for crop protection: knocking out of susceptibility genes, introduction of resistance genes, and modulation of defence genes. Potential applications of CRISPR/Cas9 in crop protection include the introduction of genes conferring resistance to pathogens, disruption of insect genes responsible for survival and reproduction and engineering of herbicide-resistant crops. In conclusion, CRISPR/Cas9 holds great promises in advancing crop protection and thus ensuring food security amidst environmental and population pressures. This review highlights the transformative potential of genome editing in crop protection and calls for continued research and development in this field. [ABSTRACT FROM AUTHOR]

Soil salinity is a major threat to global agriculture, limiting plant growth and lowering crop yields. Recent advances in CRISPR/Cas9 genome editing technology provide unprecedented precision and efficiency for addressing these challenges by directly modifying the central dogma (CD) of molecular biology in plants. The CD naturally lends itself to tighter multi-level regulation, where transcription and translation are both under control at the same time. A multilayer component of CD such as epigenetic modification, transcription, post-transcriptional modification, translation, and post-translational modification contributes significantly to stress tolerance. Strict control of CD components by Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-associated protein 9 (CRISPR/Cas9) might lead to the generation of climate smart crops. This review delves into the latest developments in the CRISPR toolbox that improve plant salt tolerance. By targeting key genes involved in transcription and translation, CRISPR/Cas9 makes it easier to modify critical components of the central dogma, allowing plants to better manage salt stress. We explore various CRISPR-based strategies, including base editing, prime editing, transcription regulation, multiplexing, RNA and many more, that reprogram gene expression and protein function to improve salt tolerance. In addition, we discuss how CRISPR can be combined with transcriptional regulation and epigenetic modifications to provide a comprehensive approach to salinity resistance for plants. The review also addresses the issues of off-target effects and efficient delivery systems, recommending novel solutions to improve the precision and applicability of CRISPR technology. This review emphasizes the transformative potential of CRISPR in modifying the central dogma to develop salt-tolerant crops, thereby contributing to sustainable agriculture and global food security. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) are known to exhibit various beneficial roles in fermentation, serving as probiotics, and producing a plethora of valuable compounds including antimicrobial activity such as bacteriocin-like inhibitory substance (BLIS) that can be used as biopreservative to improve food safety and quality. However, the yield of BLIS is often limited, which poses a challenge to be commercially competitive with the current preservation practice. Therefore, the present work aimed to establish an optimised two-plasmid CRISPR/Cas9 system to redirect the carbon flux away from lactate towards compounds with antimicrobial activity by disrupting lactate dehydrogenase gene (ldh) on various strains of LAB. The lactic acid-deficient (ldhΔ) strains caused a metabolic shift resulting in increased inhibitory activity against selected foodborne pathogens up to 78 % than the wild-type (WT) strain. The most significant effect was depicted by Enterococcus faecalis-ldh∆ which displayed prominent bactericidal effects against all foodborne pathogens as compared to the WT that showed no antimicrobial activity. The present work provided a framework model for economically important LAB and other beneficial bacteria to synthesise and increase the yield of valuable food and industrial compounds. The present work reported for the first time that the metabolism of selected LAB can be manipulated by modifying ldh to attain metabolites with higher antimicrobial activity. • The development of a new mutant lactic acid bacteria (LAB) strains was attained by genetically disrupt lactate dehydrogenase (ldh) gene, to eliminate the production of lactic acid. • The deletion of the ldh was achieved by introducing an efficient two-plasmid CRISPR system. • The growth rate of the mutant LAB strains show significant changes due to the deletion of ldh. • The effects of metabolic alteration on Bacteriocin-Like Inhibitory Substance (BLIS) was significantly increase up to two-fold resulted from re-routing of the carbon flow towards BLIS pathway. [ABSTRACT FROM AUTHOR]

Background: Ascorbate peroxidase (APX) has emerged as a promising target for chemotherapy because of its absence in humans and crucial role in the antioxidant defence of trypanosomatids. APXs, which are class I haeme-containing enzymes, reduces hydrogen peroxide using ascorbate to produce water and monodehydroascorbate, thereby preventing cell damage caused by H2O2. Methods: We aimed to create an APX-knockout L. infantum line using CRISPR/Cas9. Despite unsuccessful attempts at full knockouts, we achieved deletion of chromosomal copies post-APX episomal insertion, yielding LiΔchrAPX::LbAPX parasites. We performed phenotypic characterization to assess the impact of these genetic modifications, which included the determination of APX transcript expression levels using quantitative PCR, drug sensitivity, infectivity, and parasite survival in macrophages. Results: Quantitative polymerase chain reaction (PCR) analysis revealed a 10- to 13-fold reduction in APX transcript expression in LiΔchrAPX::LbAPX compared with wild-type (LiWT) and APX-overexpressing (Li::Cas9::LbAPX) parasites, respectively. The episomes in those knockdown parasites remained stable even after 20 drug-free passages in vitro. Li::Cas9::LbAPX parasites showed increased resistance to trivalent antimony (SbIII) and isoniazid, reduced tolerance to H2O2, and unchanged macrophage infectivity compared with LiWT. In contrast, LiΔchrAPX::LbAPX parasites were more sensitive to SbIII and isoniazid, exhibited greater susceptibility to H2O2-induced oxidative stress, and 72 h post-infection, showed fewer infected macrophages and intracellular amastigotes compared with LiWT parasites. Conclusions: Our findings hint at the indispensability of APX in L. infantum and raise the possibility of its potential as a therapeutic target for leishmaniasis. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major glycolytic enzyme that plays an important role in several cellular processes, including plant hormone signaling, plant development, and transcriptional regulation. In this study, we divided it into four groups through structural analysis of eight GAPDH genes identified in the rice genome. Among them, the expression level of five genes of cytosolic GAPDH was shown to be different for each organ. The mutation induction of the GAPDHC7 gene by the CRISPR/Cas9 system revealed that the 7 bp and 2 bp deletion, early end codon, was used in protein production. In addition, the selected mutants showed lower plant heights compared to the wild-type plants. To investigate the effect on carbohydrate metabolism, the expression of the genes of starch-branched enzyme I (SbeI), sucrose synthase (SS), and 3-phosphoglycer phosphokinase (PGK) increased the expression of the SBeI gene threefold in the knockout lines compared to the wild-type (WT) plant, while the expression of the SS and PGK genes decreased significantly. And the starch and soluble sugar content of the knockout lines increased by more than 60% compared to the WT plant. Also, the free amino acid content was significantly increased in the Gln and Asn contents of the knockout lines compared to the WT plants, while the contents of Gly and Ser were decreased. Our results suggest that OsGAPDHC7 has a great influence on energy metabolism, such as pre-harvested sprouting and amino acid content. [ABSTRACT FROM AUTHOR]

Lepidopteran insects are a major threat to global agriculture, causing significant crop losses and economic damage. Traditional pest control methods are becoming less effective due to the rapid evolution of insecticide resistance. This study explores the current status and genomic characteristics of 1315 Lepidopteran records, alongside an overview of relevant research, utilizing advanced functional genomics techniques, including RNA-seq and CRISPR/Cas9 gene-editing technologies to uncover the molecular mechanisms underlying insecticide resistance. Our genomic analysis revealed significant variability in genome size, assembly quality, and chromosome number, which may influence species' biology and resistance mechanisms. We identified key resistance-associated genes and pathways, including detoxification and metabolic pathways, which help these insects evade chemical control. By employing CRISPR/Cas9 gene-editing techniques, we directly manipulated resistance-associated genes to confirm their roles in resistance, demonstrating their potential for targeted interventions in pest management. These findings emphasize the value of integrating genomic data into the development of effective and sustainable pest control strategies, reducing reliance on chemical insecticides and promoting environmentally friendly integrated pest management (IPM) approaches. Our study highlights the critical role of functional genomics in IPM and its potential to provide long-term solutions to the growing challenge of Lepidopteran resistance. [ABSTRACT FROM AUTHOR]

It is estimated that nearly all individuals have been infected with herpesviruses, with herpes simplex virus type 1 (HSV-1) representing the most prevalent virus. In most cases, HSV-1 causes non-life-threatening skin damage in adults. However, in patients with compromised immune systems, it can cause serious diseases, including death. The situation is further complicated by the emergence of strains that are resistant to both traditional and novel antiviral drugs. It is, therefore, imperative that new methods of combating HSV-1 and other herpesviruses be developed without delay. CRISPR/Cas systems may prove an effective means of controlling herpesvirus infections. This review presents the current understanding of the underlying molecular mechanisms of HSV-1 infection and discusses four potential applications of CRISPR/Cas systems in the fight against HSV-1 infections. These include the search for viral and cellular genes that may serve as effective targets, the optimization of anti-HSV-1 activity of CRISPR/Cas systems in vivo, the development of CRISPR/Cas-based HSV-1 diagnostics, and the validation of HSV-1 drug resistance mutations. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) represent one of the most severe forms of genetic damage in organisms, yet vertebrate models capable of monitoring DSBs in real-time remain scarce. BRCA1/BRCA2-containing complex subunit 3 (BRCC3), also known as BRCC36, functions within various multiprotein complexes to mediate diverse biological processes. However, the physiological role of BRCC3 in vertebrates, as well as the underlying mechanisms that govern its activity, are not well understood. To explore these questions, we generated brcc3-knockout zebrafish using CRISPR/Cas9 gene-editing technology. While brcc3 mutant zebrafish appear phenotypically normal and remain fertile, they exhibit significantly increased rates of mortality and deformity following exposure to DNA damage. Furthermore, embryos lacking Brcc3 display heightened p53 signaling, elevated γ-H2AX levels, and increased apoptosis in response to DNA-damaging agents such as ultraviolet (UV) light and Etoposide (ETO). Notably, genetic inactivation of p53 or pharmacological inhibition of Ataxia-telangiectasia mutated (ATM) activity rescues the hypersensitivity to UV and ETO observed in Brcc3-deficient embryos. These findings suggest that Brcc3 plays a critical role in DNA damage response (DDR), promoting cell survival during embryogenesis. Additionally, brcc3-null mutant zebrafish offer a promising vertebrate model for real-time monitoring of DSBs. [ABSTRACT FROM AUTHOR]

In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes. [ABSTRACT FROM AUTHOR]

The cotton bollworm causes severe mechanical damage to plants during feeding and leaves oral secretions (OSs) at the mechanical wounds. The role these OSs play in the invasion of plants is still largely unknown. Here, a novel H. armigera effector peptidyl prolyl trans‐isomerase 5 (PPI5) was isolated and characterized. PPI5 induces the programmed cell death (PCD) due to the unfolded protein response (UPR) in tobacco leaf. We reveal that PPI5 is important for the growth and development of cotton bollworm on plants, as it renders plants more susceptible to feeding. The GhFKBP17‐2, was identified as a host target for PPI5 with peptidyl‐prolyl isomerase (PPIase) activity. CRISPR/Cas9 knock‐out cotton mutant (CR‐GhFKBP17‐1/3), VIGS (TRV: GhFKBP17‐2) and overexpression lines (OE‐GhFKBP17‐1/3) were created and the data indicate that GhFKBP17‐2 positively regulates endoplasmic reticulum (ER) stress‐mediated plant immunity in response to cotton bollworm infestation. We further confirm that PPI5 represses JA and SA levels by downregulating the expression of JA‐ and SA‐associated genes, including JAZ3/9, MYC2/3, JAR4, PR4, LSD1, PAD4, ICS1 and PR1/5. Taken together, our results reveal that PPI5 reduces plant defense responses and makes plants more susceptible to cotton bollworm infection by targeting and suppressing GhFKBP17‐2 ‐mediated plant immunity. [ABSTRACT FROM AUTHOR]

Genetic mutations significantly contribute to the onset of diseases, with over half of the cases caused by single‐nucleotide mutations. Advances in gene editing technologies have enabled precise editing and correction of mutated genes, offering effective treatment methods for genetic disorders. CRISPR/Cas9, despite its power, poses risks of inducing gene mutations due to DNA double‐strand breaks (DSB). The advent of base editing (BE) and prime editing (PE) has mitigated these risks by eliminating the hazards associated with DNA DSBs, allowing for more precise gene editing. This breakthrough lays a solid foundation for the clinical application of gene editing technologies. This review discusses the principles, development, and applications of PE gene editing technology in various genetic mutation‐induced diseases. [ABSTRACT FROM AUTHOR]

Choroidal neovascularization (CNV) is a leading cause of visual impairment in wet age-related macular degeneration (wAMD). Recent investigations have validated the potential of reducing glutamine synthetase (GS) to inhibit neovascularization formation, offering prospects for treating various neovascularization-related diseases. In this study, we devised a CRISPR/Cas9 delivery system employing the nucleic acid aptamer AS1411 as a targeting moiety and exosome-liposome hybrid nanoparticles as carriers (CAELN). Exploiting the binding affinity between AS1411 and nucleolin on endothelial cell surfaces, the delivery system was engineered to specifically target the glutamine synthetase gene (GLUL), thereby attenuating GS levels and continuously suppressing CNV. CAELN exhibited spherical and uniform dispersion. In vitro cellular investigations demonstrated gene editing efficiencies of CAELN ranging from 42.05 to 55.02% and its capacity to inhibit neovascularization in HUVEC cells. Moreover, in vivo pharmacodynamic studies conducted in CNV rabbits revealed efficacy of CAELN in restoring the thickness of intra- and extranuclear tissues. The findings suggest that GS is a novel target for the inhibition of pathological CNV, while the development of AS1411-modified exosome-liposome hybrid nanoparticles represents a novel delivery method for the treatment of neovascular-related diseases. [ABSTRACT FROM AUTHOR]

Chlamydomonas reinhardtii, a model organism for unicellular green microalgae, is widely used in basic and applied research. Nonetheless, proceeding towards synthetic biology requires a full set of manipulation techniques for inserting, removing, or editing genes. Despite recent advancements in CRISPR/Cas9, still significant limitations in producing gene knock-outs are standing, including (i) unsatisfactory genome editing (GE) efficiency and (ii) uncontrolled DNA random insertion of antibiotic resistance markers. Thus, obtaining efficient gene targeting without using marker genes is instrumental in developing a pipeline for efficient engineering of strains for biotechnological applications. We developed an efficient DNA-free gene disruption strategy, relying on phenotypical identification of mutants, to (i) precisely determine its efficiency compared to marker-relying approaches and (ii) establish a new DNA-free editing tool. This study found that classical CRISPR Cas9-based GE for gene disruption in Chlamydomonas reinhardtii is mainly limited by DNA integration. With respect to previous results achieved on synchronized cell populations, we succeeded in increasing the GE efficiency of single gene targeting by about 200 times and up to 270 times by applying phosphate starvation. Moreover, we determined the efficiency of multiplex simultaneous gene disruption by using an additional gene target whose knock-out did not lead to a visible phenotype, achieving a co-targeting efficiency of 22%. These results expand the toolset of GE techniques and, additionally, lead the way to future strategies to generate complex genotypes or to functionally investigate gene families. Furthermore, the approach provides new perspectives on how GE can be applied to (non-) model microalgae species, targeting groups of candidate genes of high interest for basic research and biotechnological applications. [ABSTRACT FROM AUTHOR]

The intensifying challenges posed by global climate change and water scarcity necessitate enhancements in agricultural productivity and sustainability within arid regions. This review synthesizes recent advancements in genetic engineering, molecular breeding, precision agriculture, and innovative water management techniques aimed at improving crop drought resistance, soil health, and overall agricultural efficiency. By examining cutting-edge methodologies, such as CRISPR/Cas9 gene editing, marker-assisted selection (MAS), and omics technologies, we highlight efforts to manipulate drought-responsive genes and consolidate favorable agronomic traits through interdisciplinary innovations. Furthermore, we explore the potential of precision farming technologies, including the Internet of Things (IoT), remote sensing, and smart irrigation systems, to optimize water utilization and facilitate real-time environmental monitoring. The integration of genetic, biotechnological, and agronomic approaches demonstrates a significant potential to enhance crop resilience against abiotic and biotic stressors while improving resource efficiency. Additionally, advanced irrigation systems, along with soil conservation techniques, show promise for maximizing water efficiency and sustaining soil fertility under saline–alkali conditions. This review concludes with recommendations for a further multidisciplinary exploration of genomics, sustainable water management practices, and precision agriculture to ensure long-term food security and sustainable agricultural development in water-limited environments. By providing a comprehensive framework for addressing agricultural challenges in arid regions, we emphasize the urgent need for continued innovation in response to escalating global environmental pressures. [ABSTRACT FROM AUTHOR]

Cuproptosis, a novel copper‐driven cell death, offers potential for cancer therapy, yet challenges persist in targeted delivery of copper ions and therapeutic resistance management. Here, CuTG‐Cas9@PLL, a cuproptosis nano‐inducer for efficient delivery of Cu ions, 6‐Thio‐dG (6‐thio‐2′‐deoxyguanosine), and CRISPR/Cas9 to enhance cuproptosis and immunotherapy via telomere stress and metabolic interference is developed. This system, combined with microneedle patches for transdermal delivery in melanoma treatment, targets the “Warburg effect,” a key resistance mechanism, through CRISPR/Cas9‐mediated LDHA knockout, thereby sensitizing cancer cells to cuproptosis and reversing immune suppression. Moreover, 6‐Thio‐dG‐induced telomere stress not only amplifies cuproptosis via autophagy dysfunction but also boosts anti‐tumor immunity by increased immunogenicity of senescent cancer cells. This work presents a novel approach for developing efficient cuproptosis nano‐inducers and reports on the feasibility of enhancing cancer cuproptosis and anti‐tumor immunotherapy through telomere stress induction and CRISPR/Cas9‐mediated metabolic interference. [ABSTRACT FROM AUTHOR]

Melanin plays a pivotal role in insect body pigmentation, significantly contributing to their adaptation to diverse biotic and abiotic environmental challenges. Several genes involved in insect melanin synthesis showed pleiotropic effects on insect development and reproduction. Among these, the N‐β‐alanyl dopamine synthetase gene (Ebony) is integral to the pigmentation process. However, the full spectrum of its pleiotropic impacts is not yet thoroughly understood. In this study, we identified and characterised the HaEbony gene in the Asian multi‐coloured ladybird beetle (Harmonia axyridis) and found that HaEbony gene is a conserved gene within the Coleoptera order. We aimed to further explore the multiple roles of HaEbony in the physiology and behaviour in H. axyridis. The CRISPR/Cas9 system was applied to generate multiple HaEbony knockout allele (HaEbony+/−), showing nucleotide deletion in the G0 and G1 generations. Remarkably, the resultant HaEbony+/− mutants consistently displayed darker pigmentation than their wild‐type counterparts across larval, pupal and adult stages. Furthermore, these HaEbony+/− individuals (G0) demonstrated an enhanced predatory efficiency, evidenced by a higher number of aphids consumed compared to the wild type. A significant finding was the reduced egg hatchability in both G0 and G1 generations of the HaEbony+/− group, highlighting a potential reproductive fitness cost associated with HaEbony deficiency. In conclusion, our study not only sheds light on the multifaceted roles of HaEbony in H. axyridis but also highlights the potential of employing CRISPR/Cas9‐targeted modifications of the Ebony gene. Such genetic interventions could enhance the environmental adaptability and predatory efficacy of ladybirds, presenting a novel strategy in biological control application. [ABSTRACT FROM AUTHOR]

Improving the efficiency of germplasm innovation has always been the aim of rice breeders. Traditional hybrid breeding methods for variety selection rarely meet the practical needs of rice production. The emergence of genome-editing technologies, such as CRISPR/Cas9, provides a new approach to the genetic improvement of crops such as rice. The number of published scientific papers related to "gene editing" and "CRISPR/Cas9" retrievable on websites both from China and other countries exhibited an increasing trend, year by year, from 2014 to 2023. Research related to gene editing in rice accounts for 33.4% and 12.3% of all the literature on gene editing published in China and other countries, respectively, much higher than that on maize and wheat. This article reviews recent research on CRISPR/Cas9 gene-editing technology in rice, especially germplasm innovation and genetic improvement of commercially promoted varieties with improved traits such as disease, insect, and herbicide resistance, salt tolerance, quality, nutrition, and safety. The aim is to provide a reference for the precise and efficient development of new rice cultivars that meet market demand. [ABSTRACT FROM AUTHOR]

Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient's dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient's fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient's skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts. [ABSTRACT FROM AUTHOR]

Pigs have long been integral to human society for their roles in agriculture and medicine. Consequently, there is an urgent need for genetic improvement of pigs to meet human dual needs for medicine and food. In agriculture, gene editing can improve productivity traits, such as growth rate and disease resistance, which could lower farming costs and benefit consumers through enhanced meat quality. In biomedical research, gene-edited pigs offer invaluable resources as disease models and in xenotransplantation, providing organs compatible with human physiology. Currently, with CRISPR technology, especially the CRISPR/Cas9 system emerging as a transformative force in modern genetics, pigs are not only sources of sustenance but also cornerstones of biomedical innovation. This review aims to summarize the applications of CRISPR/Cas9 technology in developing pigs that serve dual roles in agriculture and biomedical applications. Compared to ZFNs and TALENs, the CRISPR/Cas9 system offers several advantages, including higher efficiency, greater specificity, ease of design and implementation, and the capability to target multiple genes simultaneously, significantly streamlining the process of genetic modifications in complex genomes. Therefore, CRISPR technology supports the enhancement of traits beneficial for agricultural productivity and facilitates applications in medicine. Furthermore, we must acknowledge the inherent deficiencies and technical challenges of the CRISPR/Cas9 technology while also anticipating emerging technologies poised to surpass CRISPR/Cas9 as the next milestones in gene editing. We hypothesize that with the continuous advancements in gene editing technologies and successful integration of traits beneficial to both agricultural productivity and medical applications, the goal of developing dual-purpose pigs for both agricultural and medical use can ultimately be achieved. [ABSTRACT FROM AUTHOR]

Y box-binding protein 1 (Ybx1/ybx1) regulates gene expression through DNA/RNA binding. In zebrafish, Ybx1 is highly abundant in primary growth (PG) follicles in the ovary, but decreases precipitously as the follicles enter the secondary growth (SG). To understand Ybx1 function in folliculogenesis, we created a ybx1 mutant using TALEN and observed disrupted folliculogenesis during the previtellogenic (PV) to early vitellogenic (EV) transition of SG, resulting in underdeveloped ovaries and infertility. Expression and western blot analyses revealed differential gene expression between ybx1-/- and control ovaries, with significantly increased expression of cdkn1a (p21), a cell cycle inhibitor, in ybx1-/- follicles. While cdkn1a knockout via CRISPR/Cas9 was embryonically lethal, the heterozygote (cdkn1a+/-) displayed advanced follicle activation and maturation, contrasting with the ybx1-/- phenotype. Partial loss of p21 alleviated the ybx1-/- phenotype, restoring folliculogenesis with normal PG-PV and PV-EV transitions in ybx1-/-;cdkn1a+/- mutants. While ybx1-/- mutant follicle cells displayed poor proliferation in vivo and in vitro, the cells from the ybx1-/-;cdkn1a+/- follicles resumed normal proliferation. In conclusion, Ybx1 is crucial for early folliculogenesis in zebrafish, potentially by repressing cdkn1a expression, either directly or indirectly. [ABSTRACT FROM AUTHOR]

A electrochemical biosensor was designed utilizing a CRISPR Cas9n-driven DNA walker combined with gold-nanosphere-like covalent organic frameworks (COFs-AuNPs) to detect breast cancer markers (PIK3CA E545K ctDNA). The DNA walker probe is activated only in the presence of circulating tumor deoxyribonucleic acid (ctDNA), binding to a support probe to form a double strand that is then specifically cleaved by the Cas9n/sgRNA complex. This cleavage produces numerous DNA fragments for signal amplification. The COF-AuNPs as electrode materials facilitate electronic transfer and provide additional active sites for the immobilization of nucleic acid probes. This setup achieves a detection limit of 1.76 aM, demonstrating high sensitivity. Additionally, Cas9n improves the specificity of the sensor, accurately distinguishing a pair of base-mismatched sequences, and reducing the occurrence of false positives. Overall, the sensor exhibits excellent selectivity, reproducibility, and potential for early diagnosis of breast cancer. [ABSTRACT FROM AUTHOR]

Myostatin is a member of the TGF-ß superfamily and functions as a negative regulator for skeletal muscle development and growth. It has become the most targeted gene in aquaculture that used for selective breeding. Previous studies involved in genome editing in several fish species confirmed that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system was highly efficient with lower off-target effect, however, no reports were raised in Culter alburnus. In this study, we employed CRISPR/Cas9 gene editing system to successfully disrupt mstn gene by co-injection with Cas9 protein and the targeted sgRNA in C. alburnus. Various Indel mutations were obtained with 82% knockout efficiency in the F0 generation by PCR sequencing. In addition, mutations in mstn that induced by CRISPR/Cas9 were detected in the F1 generation by individually mating the wild-type female with the F0 generation of mstn-KO male at sexual maturity. More importantly, the body weight and length were significantly elevated in mstn ± group when compared to those of the control. As expected in mstn ± group, the expression level of mstn was sharply reduced, whereas a slight increase was observed in two growth-related genes (myod and myog). Moreover, higher numbers of muscle fibers were observed in mstn ± group, meaning that growth performance in mstn ± individuals might be represented by increasing the number of muscle fibers. Taken together, our current study successfully obtained a site-specific modification of mstn using CRISPR/Cas9 technology, and these results provided a new insight for facilitating topmouth culter genetic studies and breeding. [ABSTRACT FROM AUTHOR]

Forests worldwide are under increasing pressure from climate change and emerging diseases, threatening their vital ecological and economic roles. Traditional breeding approaches, while valuable, are inherently slow and limited by the long generation times and existing genetic variation of trees. CRISPR technologies offer a transformative solution, enabling precise and efficient genome editing to accelerate the development of climate-resilient and productive forests. This review provides a comprehensive overview of CRISPR applications in forestry, exploring its potential for enhancing disease resistance, improving abiotic stress tolerance, modifying wood properties, and accelerating growth. We discuss the mechanisms and applications of various CRISPR systems, including base editing, prime editing, and multiplexing strategies. Additionally, we highlight recent advances in overcoming key challenges such as reagent delivery and plant regeneration, which are crucial for successful implementation of CRISPR in trees. We also delve into the potential and ethical considerations of using CRISPR gene drive for population-level genetic alterations, as well as the importance of genetic containment strategies for mitigating risks. This review emphasizes the need for continued research, technological advancements, extensive long-term field trials, public engagement, and responsible innovation to fully harness the power of CRISPR for shaping a sustainable future for forests. [ABSTRACT FROM AUTHOR]

Watermelon is one of the most important cucurbit crops, but its production is seriously affected by viral infections. Although eIF4E proteins have emerged as the major mediators of the resistance to viral infections, the mechanism underlying the contributions of eIF4E to watermelon disease resistance remains unclear. In this study, three CleIF4E genes and one CleIF(iso)4E gene were identified in the watermelon genome. Among these genes, CleIF4E1 was most similar to other known eIF4E genes. To investigate the role of CleIF4E1, CRISPR/Cas9 technology was used to knock out CleIF4E1 in watermelon. One selected mutant line had an 86 bp deletion that resulted in a frame-shift and the expression of a truncated protein. The homozygous mutant exhibits developmental defects in plant growth, leaf morphology and reduced yield. Furthermore, the mutant was protected against the zucchini yellow mosaic virus, but not the cucumber green mottled mosaic virus. In summary, this study preliminarily clarified the functions of eIF4E proteins in watermelon. The generated data will be useful for elucidating eIF4E-related disease resistance mechanisms in watermelon. The tissue-specific editing of CleIF4E1 in future studies may help to prevent adverse changes to watermelon fertility. [ABSTRACT FROM AUTHOR]

Summary DNA methylation is an essential epigenetic modification for gene regulation in plant growth and development. However, the precise mechanisms of DNA methylation remain poorly understood, especially in woody plants. We employed whole‐genome bisulfite sequencing (WGBS), assays for transposase‐accessible chromatin using sequencing (ATAC‐seq), and RNA‐Seq to investigate epigenetic regulatory relationships in Populus tomentosa treated with DNA methylation inhibitor 5‐azacitidine. Expression‐quantitative trait methylation analysis (eQTM), epigenome‐wide association study (EWAS), and joint linkage‐linkage disequilibrium mapping were used to explore the epigenetic regulatory genes, and using CRISPR/Cas9 to identify the role of candidate genes. Plant developmental abnormalities occurred when DNA methylation levels were substantially reduced. DNA methylation regulated 112 expressed genes via chromatin accessibility, of which 61 genes were significantly influenced by DNA methylation variation at the population level. One DNA methylation‐regulated gene, PtoGntK, was located in a major quantitative trait locus (QTL) for poplar growth. Overexpression and CRISPR/Cas9 of PtoGntK revealed it affected poplar height and stem diameter. The PtoRAP2.12 was found to bind to the demethylated accessible region in the PtoGntK promoter, thereby promoting growth in poplar. This study identified key genes with epigenetic regulation for plant growth and provides insights into epigenetic regulation mechanisms in woody plants. [ABSTRACT FROM AUTHOR]

SUMMARY Manipulating plant height is an essential component of crop improvement. Plant height was generally reduced through breeding in wheat, rice, and sorghum to resist lodging and increase grain yield but kept high for bioenergy crops. Here, we positionally cloned a plant height quantitative trait locus (QTL) qHT7.1 as a MYB transcription factor controlling internode elongation, cell proliferation, and cell morphology in sorghum. A 740 bp transposable element insertion in the intronic region caused a partial mis‐splicing event, generating a novel transcript that included an additional exon and a premature stop codon, leading to short plant height. The dominant allele had an overall higher expression than the recessive allele across development and internode position, while both alleles' expressions peaked at 46 days after planting and progressively decreased from the top to lower internodes. The orthologue of qHT7.1 was identified to underlie the brachytic1 (br1) locus in maize. A large insertion in exon 3 and a 160 bp insertion at the promoter region were identified in the br1 mutant, while an 18 bp promoter insertion was found to be associated with reduced plant height in a natural recessive allele. CRISPR/Cas9–induced gene knockout of br1 in two maize inbred lines showed significant plant height reduction. These findings revealed functional connections across natural, mutant, and edited alleles of this MYB transcription factor in sorghum and maize. This enriched our understanding of plant height regulation and enhanced our toolbox for fine‐tuning plant height for crop improvement. [ABSTRACT FROM AUTHOR]

It is still difficult to manipulate the nucleotide-binding site-leucine-rich repeat (NB-LRR) class disease resistance genes because of their large multigenic family. Here, we report the successful application of CRISPR/Cas9 to resurrect a functional allele from a susceptible allele of the N′ tobamovirus resistance gene. The susceptible alleles of N′ from some Nicotiana tabacum cultivars (Nt-n′) have a partial duplication of the N′ coding sequence upstream of a complete coding sequence of the N′ gene, which likely abolished the N′-mediated resistance. We first established a transgenic tobacco line expressing Cas9 under the control of a chemically inducible promoter. The plant line was retransformed with a construct expressing a guide RNA targeting the sequences common to the duplicated partial sequence in the upstream and the complete sequence in the downstream. The T0 transformants had different ratios of the sequences devoid of the duplicated partial sequence. Sequencing proved that some of them had sequences identical to that of the functional N′ gene, suggesting the successful resurrection of the functional N′ gene. The resurrected allele, N′-R, was inherited by a few T1 progenies and subsequent generations with the least mutation at the target site under Cas9-uninduced conditions. The plants homozygous for N′-R showed resistance to a tobamovirus, indicating that the resurrected N′-R allele is functional. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA), a type of sesquiterpenoid plant hormone, has high application value in agriculture, nutrition and medicine. Herein, we constructed an efficient ABA-producing yeast cell factory by combining multidimensional engineering strategies. Starting from a suitable strain, YS010 was selected from 11 varieties of S. cerevisiae strains by evaluating ergosterol content and growth ability, then the biosynthetic pathway of ABA derived from Botris cinerea was constructed, resulting in 1.93 mg L−1 ABA. Next, the metabolic flux of the mevalonic acid (MVA) pathway was increased to enhance the synthesis of the precursor farnesyl pyrophosphate (FPP). To further increase the FPP competitiveness of the ABA synthesis pathway, we attempted to enhance the catalytic performance of BcABA3 through enzyme engineering, and the ABA yield of the mutant strain YS036-ABAPA206D reached 2.64 mg L−1 in SC-ura medium. In addition, we developed a multi-copy integration strategy, TPI1-delta driven CRISPR-Cas9 (TDI-CRISPR) integration system, to realize the high copy and stable expression of bcaba1 , bcaba2 and bcaba3 , which enabled the titer of ABA to reach 17.47 mg L−1. Finally, by optimising the fermentation medium, the ABA titer reached 30.30 mg L−1, which was the highest level ever reported for de novo ABA biosynthesis in S. cerevisiae. [Display omitted] • The engineered strain produced 30.30 mg L−1 of abscisic acid (ABA). • Rational design of α-ionylideneethane synthase (BcABA3) improved its activity. • ABA production significantly increased using the developed integration system. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats associated with protein 9) was first identified as a component of the bacterial adaptive immune system and subsequently engineered into a genome-editing tool. The key breakthrough in this field came with the realization that CRISPR/Cas9 could be used in mammalian cells to enable transformative genetic editing. This technology has since become a vital tool for various genetic manipulations, including gene knockouts, knock-in point mutations, and gene regulation at both transcriptional and post-transcriptional levels. CRISPR/Cas9 holds great potential in human medicine, particularly for curing genetic disorders. However, despite significant innovation and advancement in genome editing, the technology still possesses critical limitations, such as off-target effects, immunogenicity issues, ethical considerations, regulatory hurdles, and the need for efficient delivery methods. To overcome these obstacles, efforts have focused on creating more accurate and reliable Cas9 nucleases and exploring innovative delivery methods. Recently, functional biomaterials and synthetic carriers have shown great potential as effective delivery vehicles for CRISPR/Cas9 components. In this review, we attempt to provide a comprehensive survey of the existing CRISPR-Cas9 delivery strategies, including viral delivery, biomaterials-based delivery, synthetic carriers, and physical delivery techniques. We underscore the urgent need for effective delivery systems to fully unlock the power of CRISPR/Cas9 technology and realize a seamless transition from benchtop research to clinical applications. [ABSTRACT FROM AUTHOR]

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Gene mutation correction was challenging until the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas). CRISPR is a new era for genome modification, and this technology has bypassed the limitations of previous methods such as zinc-finger nuclease and transcription activator-like effector nuclease. Currently, this method is becoming the method of choice for gene-editing purposes, especially therapeutic gene editing in diseases such as cardiovascular, neurological, renal, genetic, optical, and stem cell, as well as blood disorders and muscular degeneration. However, finding the optimum delivery system capable of carrying this large complex persists as the main challenge of this technology. Therefore, it would be ideal if the delivery vehicle could direct the introduction of editing functions to specific cells in a multicellular organism. Exosomes are membrane-bound vesicles with high biocompatibility and low immunogenicity; they offer the best and most reliable way to fill the CRISPR/Cas9 system delivery gap. This review presents the current evidence on the molecular mechanisms and challenges of CRISPR/Cas9-mediated genome modification. Also, the role of CRISPR/Cas9 in the development of treatment and diagnosis of numerous disorders, from malignancies to viral infections, has been discussed. Lastly, the focus is on new advances in exosome-delivery technologies that may play a role in CRISPR/Cas9 delivery for future clinical settings. [ABSTRACT FROM AUTHOR]

Strategies to enhance rice productivity in response to global demand have been the paramount focus of breeders worldwide. Multiple factors, including agronomical traits such as plant architecture and grain formation and physiological traits such as photosynthetic efficiency and NUE (nitrogen use efficiency), as well as factors such as phytohormone perception and homeostasis and transcriptional regulation, indirectly influence rice grain yield. Advances in genetic analysis methodologies and functional genomics, numerous genes, QTLs (Quantitative Trait Loci), and SNPs (Single-Nucleotide Polymorphisms), linked to yield traits, have been identified and analyzed in rice. Genome editing allows for the targeted modification of identified genes to create novel mutations in rice, avoiding the unintended mutations often caused by random mutagenesis. Genome editing technologies, notably the CRISPR/Cas9 system, present a promising tool to generate precise and rapid modifications in the plant genome. Advancements in CRISPR have further enabled researchers to modify a larger number of genes with higher efficiency. This paper reviews recent research on genome editing of yield-related genes in rice, discusses available gene editing tools, and highlights their potential to expedite rice breeding programs. [ABSTRACT FROM AUTHOR]

Changes in climatic conditions increase the frequency of severity caused by abiotic stress. Understanding the physiological responses to abiotic stress is crucial for developing action plans to increase stress tolerance in plants, whether through classical breeding, genetic engineering, or other innovative approaches. Gene editing in plants is a quickly advancing field that involves the targeted modification of plant genomes to achieve specific traits or characteristics. One of the plants' most extensively used gene-editing technologies is Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR-Cas9). CRISPR-Cas9 allows making precise changes to the DNA of plants by introducing targeted mutations. Efforts to address these challenges involve the development of stress-tolerant plant varieties through breeding, genetic engineering, and gene editing. These approaches aim to increase the ability of plants to withstand and recover from abiotic stress, ultimately improving crop resilience, quality, and yield in challenging environments. Additionally, sustainable agricultural practices and precision farming techniques can be employed to optimize resource use and mitigate the impact of abiotic stresses on crop production. [ABSTRACT FROM AUTHOR]