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During the past 20 years, gene editing has emerged as a novel form of gene therapy. Since the publication of the first potentially therapeutic gene editing platform for genetic disorders, increasingly sophisticated editing technologies have been developed. As with viral vector-mediated gene addition, inborn errors of immunity are excellent candidate diseases for a corrective autologous hematopoietic stem cell gene editing strategy. Research on gene editing for inborn errors of immunity is still entirely preclinical, with no trials yet underway. However, with editing techniques maturing, scientists are investigating this novel form of gene therapy in context of an increasing number of inborn errors of immunity. Here, we present an overview of these studies and the recent progress moving these technologies closer to clinical benefit.

A tool for precise, target-specific, efficient, and affordable genome editing is a dream for many researchers, from those who conduct basic research to those who use it for applied research. Since 2012, we have tool that almost fulfils such requirements; it is based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes , and highlight key factors affecting its mutagenesis outcomes: (i) factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state, or Cas9 variant, and how long it remains in place after cleavage; and (ii) factors affecting the follow-up DNA repair mechanisms including mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts, and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines. [ABSTRACT FROM AUTHOR]

Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, β-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (−/−) heterozygous, bi-allelic (−/−) homozygous, and mono-allelic (−/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (−/−) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk. [ABSTRACT FROM AUTHOR]

The delivery of CRISPR/Cas systems holds immense potential for revolutionizing cancer treatment, with recent advancements focusing on extracellular vesicles (EVs) and viral vectors. EVs, particularly exosomes, offer promising opportunities for targeted therapy due to their natural cargo transport capabilities. Engineered EVs have shown efficacy in delivering CRISPR/Cas components to tumor cells, resulting in inhibited cancer cell proliferation and enhanced chemotherapy sensitivity. However, challenges such as off-target effects and immune responses remain significant hurdles. Viral vectors, including adeno-associated viruses (AAVs) and adenoviral vectors (AdVs), represent robust delivery platforms for CRISPR/Cas systems. AAVs, known for their safety profile, have already been employed in clinical trials for gene therapy, demonstrating their potential in cancer treatment. AdVs, capable of infecting both dividing and non-dividing cells, offer versatility in CRISPR/Cas delivery for disease modeling and drug discovery. Despite their efficacy, viral vectors present several challenges, including immune responses and off-target effects. Future directions entail refining delivery systems to enhance specificity and minimize adverse effects, heralding personalized and effective CRISPR/Cas-mediated cancer therapies. This article underscores the importance of optimized delivery mechanisms in realizing the full therapeutic potential of CRISPR/Cas technology in oncology. As the field progresses, addressing these challenges will be pivotal for translating CRISPR/Casmediated cancer treatments from bench to bedside. [ABSTRACT FROM AUTHOR]

The invention of next-generation CRISPR/Cas gene editing tools, like base and prime editing, for correction of gene variants causing disease, has created hope for in vivo use in patients leading to wider clinical translation. To realize this potential, delivery vehicles that can ferry gene editing tool kits safely and effectively into specific cell populations or tissues are in great demand. In this review, we describe the development of enveloped retrovirus-derived particles as carriers of "ready-to-work" ribonucleoprotein complexes consisting of Cas9-derived editor proteins and single guide RNAs. We present arguments for adapting viruses for cell-targeted protein delivery and describe the status after a decade-long development period, which has already shown effective editing in primary cells, including T cells and hematopoietic stem cells, and in tissues targeted in vivo, including mouse retina, liver, and brain. Emerging evidence has demonstrated that engineered virus-derived nanoparticles can accommodate both base and prime editors and seems to fertilize a sprouting hope that such particles can be further developed and produced in large scale for therapeutic applications. [ABSTRACT FROM AUTHOR]

Gene expression levels in rice (Oryza sativa L.) and other plant species are determined by the promoters, which directly control phenotypic characteristics. As essential components of genes, promoters regulate the intensity, location, and timing of gene expression. They contain numerous regulatory elements and serve as binding sites for proteins that modulate transcription, including transcription factors and RNA polymerases. Genome editing can alter promoter sequences, thereby precisely modifying the expression patterns of specific genes, and ultimately affecting the morphology, quality, and resistance of rice. This paper summarizes research on rice promoter editing conducted in recent years, focusing on improvements in yield, heading date, quality, and disease resistance. It is expected to inform the application of promoter editing and encourage further research and development in crop genetic improvement with promote. [ABSTRACT FROM AUTHOR]

CRISPR-based (Clustered regularly interspaced short palindromic repeats-based) technologies have revolutionized molecular biology and diagnostics, offering unprecedented precision and versatility. However, challenges remain, such as high costs, demanding technical expertise, and limited quantification capabilities. To overcome these limitations, innovative microfluidic platforms are emerging as powerful tools for enhancing CRISPR diagnostics. This review explores the exciting intersection of CRISPR and microfluidics, highlighting their potential to revolutionize healthcare diagnostics. By integrating CRISPR's specificity with microfluidics' miniaturization and automation, researchers are developing more sensitive and portable diagnostic tools for a range of diseases. These microfluidic devices streamline sample processing, improve diagnostic performance, and enable point-of-care applications, allowing for rapid and accurate detection of pathogens, genetic disorders, and other health conditions. The review discusses various CRISPR/Cas systems, including Cas9, Cas12, and Cas13, and their integration with microfluidic platforms. It also examines the advantages and limitations of these systems, highlighting their potential for detecting DNA and RNA biomarkers. The review also explores the key challenges in developing and implementing CRISPR-driven microfluidic diagnostics, such as ensuring robustness, minimizing cross-contamination, and achieving robust quantification. Finally, it highlights potential future directions for this rapidly evolving field, emphasizing the transformative potential of these technologies for personalized medicine and global health. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases (CVDs) are the leading cause of global mortality among non-communicable diseases. Current cardiac regeneration treatments have limitations and may lead to adverse reactions. Hence, innovative technologies are needed to address these shortcomings. Messenger RNA (mRNA) emerges as a promising therapeutic agent due to its versatility in encoding therapeutic proteins and targeting "undruggable" conditions. It offers low toxicity, high transfection efficiency, and controlled protein production without genome insertion or mutagenesis risk. However, mRNA faces challenges such as immunogenicity, instability, and difficulty in cellular entry and endosomal escape, hindering its clinical application. To overcome these hurdles, lipid nanoparticles (LNPs), notably used in COVID-19 vaccines, have a great potential to deliver mRNA therapeutics for CVDs. This review highlights recent progress in mRNA-LNP therapies for CVDs, including Myocardial Infarction (MI), Heart Failure (HF), and hypercholesterolemia. In addition, LNP-mediated mRNA delivery for CAR T-cell therapy and CRISPR/Cas genome editing in CVDs and the related clinical trials are explored. To enhance the efficiency, safety, and clinical translation of mRNA-LNPs, advanced technologies like artificial intelligence (AGILE platform) in RNA structure design, and optimization of LNP formulation could be integrated. We conclude that the strategies to facilitate the extra-hepatic delivery and targeted organ tropism of mRNA-LNPs (SORT, ASSET, SMRT, and barcoded LNPs) hold great prospects to accelerate the development and translation of mRNA-LNPs in CVD treatment. [Display omitted] [ABSTRACT FROM AUTHOR]

Introduction: Colon cancer, ranked as the fourth leading global cause of cancer death, exhibits a complex progression marked by genetic variations. Over the past decade, the utilization of diverse CRISPR systems has propelled accelerated research into colorectal cancer (CRC) treatment. Areas covered: CRISPR/Cas9, a key player in this research, identifies new oncogenes, tumor suppressor genes (TSGs), and drug-resistance genes. Additionally, it facilitates the construction of experimental models, conducts genome-wide library screening, and develops new therapeutic targets, especially for targeted knockout in vivo or molecular targeted drug delivery, contributing to personalized treatments and significantly enhancing the care of colon cancer patients. In this review, we provide insights into the mechanism of the CRISPR/Cas9 system, offering a comprehensive exploration of its applications in CRC, spanning screening, modeling, gene functions, diagnosis, and gene therapy. While acknowledging its transformative potential, the article highlights the challenges and limitations of CRISPR systems. Expert opinion: The application of CRISPR/Cas9 in CRC research provides a promising avenue for personalized treatments. Its potential for identifying key genes and enabling experimental models and genome-wide screening enhances patient care. This review underscores the significance of CRISPR-Cas9 gene editing technology across basic research, diagnosis, and the treatment landscape of colon cancer. [ABSTRACT FROM AUTHOR]

Niemann–Pick disease type C (NPC) is a rare neurodegenerative condition resulted from mutations in NPC1 and NPC2 genes. This cellular lipid transferring disorder mainly involves endocytosed cholesterol trafficking. The accumulation of cholesterol and glycolipids in late endosomes and lysosomes results in progressive neurodegeneration and death. Recently, genome editing technologies, particularly CRISPR/Cas9 have offered the opportunity to create disease models to screen novel therapeutic options for this disorder. Moreover, these methods have been used for the purpose of gene therapy. This review summarizes the studies that focused on the application of CRISPR/Cas9 technology for exploring the mechanism of intracellular cholesterol transferring, and screening of novel agents for treatment of NPC. [ABSTRACT FROM AUTHOR]

The methylotrophic yeast Pichia pastoris is garnering interest as a chassis cell factory for the manufacture of recombinant proteins because it effectively satisfies the requirements of both laboratory and industrial set up. The optimisation of P. pastoris cultivation is still necessary due to strain- and product-specific problems such as promoter strength, methanol utilisation type, and culturing conditions to realize the high yields of heterologous protein(s) of interest. Techniques integrating genetic and process engineering have been used to overcome these problems. Insight into the Pichia as an expression system utilizing MUT pathway and the development of methanol free systems are highlighted in this systematic review. Recent developments in the improved production of proteins in P. pastoris by (i) diverse genetic engineering such as codon optimization and gene dosage; (ii) cultivating tactics including co-expression of chaperones; (iii) advances in the use of the 2A peptide system, and (iv) CRISPR/Cas technologies are widely discussed. We believe that by combining these strategies, P. pastoris will become a formidable platform for the production of high value therapeutic proteins. [ABSTRACT FROM AUTHOR]

The development of gene editing tools has been a significant area of research in the life sciences for nearly 30 years. These tools have been widely utilized in disease detection and mechanism research. In the new century, they have shown potential in addressing various scientific challenges and saving lives through gene editing therapies, particularly in combating cardiovascular disease (CVD). The rapid advancement of gene editing therapies has provided optimism for CVD patients. The progress of gene editing therapy for CVDs is a comprehensive reflection of the practical implementation of gene editing technology in both clinical and basic research settings, as well as the steady advancement of research and treatment of CVDs. This article provides an overview of the commonly utilized DNA‐targeted gene editing tools developed thus far, with a specific focus on the application of these tools, particularly the clustered regularly interspaced short palindromic repeat/CRISPR‐associated genes (Cas) (CRISPR/Cas) system, in CVD gene editing therapy. It also delves into the challenges and limitations of current gene editing therapies, while summarizing ongoing research and clinical trials related to CVD. The aim is to facilitate further exploration by relevant researchers by summarizing the successful applications of gene editing tools in the field of CVD. [ABSTRACT FROM AUTHOR]

Climate change has made plants more vulnerable to various stresses, which presents a significant threat to the world’s food supply. Despite several efforts deployed over the last three decades in plant breeding and genetic engineering, there is still a lot to be done for crop improvement. CRISPR/Cas is a naturally occurring genome editing tool adopted from the bacterial adaptive immune defense system. Gene editing has undergone a revolutionary change since the discovery of CRISPR/Cas-based genome engineering. This emerging technology recently implemented, has enabled targeted mutagenesis with unprecedented simplicity and accuracy, making it suitable to edit DNA sequences at site-specific targets with ultimate precision. Currently, this cutting-edge technology has been extensively used and incessantly improved at several orders of magnitude to contribute as a robust approach for studying gene function with a variety of applications that can accelerate basic and applied research toward crop improvement. This mini-review briefly describes some of the major achievements in genome editing tools and highlights future outlooks for these technologies in functional genomics and applied plant biotechnology. [ABSTRACT FROM AUTHOR]

Abstract Gene expression levels in rice (Oryza sativa L.) and other plant species are determined by the promoters, which directly control phenotypic characteristics. As essential components of genes, promoters regulate the intensity, location, and timing of gene expression. They contain numerous regulatory elements and serve as binding sites for proteins that modulate transcription, including transcription factors and RNA polymerases. Genome editing can alter promoter sequences, thereby precisely modifying the expression patterns of specific genes, and ultimately affecting the morphology, quality, and resistance of rice. This paper summarizes research on rice promoter editing conducted in recent years, focusing on improvements in yield, heading date, quality, and disease resistance. It is expected to inform the application of promoter editing and encourage further research and development in crop genetic improvement with promote.

INTRODUCTION. The development of malignant neoplasms is associated with changes in the expression of small non-coding RNAs (miRNAs). This emphasises the need for research into the development of miRNA-targeted inhibitors as a promising approach to cancer treatment.AIM. This study aimed to compare current strategies for suppressing the functional activity of tumour-associated miRNAs based on the use of therapeutic nucleic acids and to determine the application potential of these strategies.DISCUSSION. This study analysed known oligonucleotide-based miRNA inhibitors with different mechanisms of action. Based on their mechanism of action, miRNA-targeted inhibitors can be classified into two groups. The first group of miRNA-targeted inhibitors exhibits an indirect inhibitory effect, either by blocking functional connections between miRNAs and specific mRNA targets through the use of miRNA-masking oligonucleotides or by introducing mutations into miRNA genes and disrupting gene biosynthesis processes through the use of the CRISPR/Cas system. Despite their relatively high biological potential, these strategies are mostly used as search tools to study miRNA functional roles and molecular interactions in carcinogenesis. The second group of oligonucleotide constructs interacts with miRNA targets directly, which leads to steric blocking or degradation of oncogenic microRNAs. These miRNA-binding oligonucleotide constructs come in a variety of structural variants, including miRNA sponges, RNA zippers, antisense oligonucleotides, and miRNases, which demonstrate high therapeutic potential in vitro and in vivo.CONCLUSION. The described analysis of the biological properties, therapeutic potential, and key advantages of the developed miRNA-targeted oligonucleotide constructs helps outline the areas for their potential practical application in cancer treatment.

Abstract Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, β-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (−/−) heterozygous, bi-allelic (−/−) homozygous, and mono-allelic (−/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (−/−) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.

SUMMARY Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium‐mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene‐editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1‐VIR2 virulence helper plasmid to improve T‐DNA delivery, and/or expressing a fusion protein between a GROWTH‐REGULATING FACTOR (GRF) and GRF‐INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1‐VIR2 helper vector with no effect on event quality regarding T‐DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5‐ to 6.5‐fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR‐/Cas9‐mediated gene editing. Our results demonstrate how a GRF‐GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene‐editing applications and molecular biology studies in maize. [ABSTRACT FROM AUTHOR]

Foodborne pathogenic infection has brought multifaceted issues to human life, leading to an urgent demand for advanced detection technologies. CRISPR/Cas-based biosensors have the potential to address various challenges that exist in conventional assays such as insensitivity, long turnaround time and complex pretreatments. In this perspective, we review the relevant strategies of CRISPR/Cas-assisted diagnostics on foodborne pathogens, focusing on biosensing platforms for foodborne pathogens based on fluorescence, colorimetric, (electro)chemiluminescence, electrochemical, and surface-enhanced Raman scattering detection. It summarizes their detection principles by the clarification of foodborne pathogenic bacteria, fungi, and viruses. Finally, we discuss the current challenges or technical barriers of these methods against broad application, and put forward alternative solutions to improve CRISPR/Cas potential for food safety. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas system has been introduced as an innovative tool for therapy, however achieving specific delivery to the target has been a major challenge. Here, an antibody‐CRISPR/Cas conjugate platform that enables specific delivery and target gene editing in HER2‐positive cancer is introduced. The CRISPR/Cas system by replacing specific residues of Cas9 with an unnatural amino acid is engineered, that can be complexed with a nanocarrier and bioorthogonally functionalized with a monoclonal antibody targeting HER2. The resultant antibody‐conjugated CRISPR/Cas nanocomplexes can be specifically delivered and induce gene editing in HER2‐positive cancer cells in vitro. It is demonstrated that the in vivo delivery of the antibody‐CRISPR/Cas nanocomplexes can effectively disrupt the plk1 gene in HER2‐positive ovarian cancer, resulting in substantial suppression of tumor growth. The current study presents a useful therapeutic platform for antibody‐mediated delivery of CRISPR/Cas for the treatment of various cancers and genetic diseases. [ABSTRACT FROM AUTHOR]

Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. miR396 genes have been shown to negatively regulate grain size in rice, but whether miR396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean miR396 genes through genome editing with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated nuclease (Cas)12SF01 in the elite soybean cultivar Zhonghuang 302 (ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants (mir396abcd and mir396acfi), and two quintuple mutants (mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfi plants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems. Taken together, our study demonstrated that loss‐of‐function of miR396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high‐yield soybean. [ABSTRACT FROM AUTHOR]

The increasing global population projected to reach 9.7 billion by 2050 from the current 7.7 billion, which is going to significantly impact food availability. Therefore, current global agricultural production needs to be increased to feed the unconstrained growing population. The changing climatic condition due to anthropogenic activities also makes the conditions more challenging to meet the required crop productivity in the future. Potato is third most consumed staple food. The current cultivation and demand of potato is particularly more in developing countries with high levels of poverty, hunger, and malnutrition because it is cheap source of nutrition and easily available. Potato is severely infected by a number of diseases, insect pests, and abiotic environmental conditions. Amidst the backdrop of climate change, the situation is deteriorating. Projections indicate that the average potato productivity in India's primary potato-growing states, responsible for approximately 90% of the nation's total potato output, is expected to decline by 2.0% in the 2050s and a more substantial 6.4% in the 2080s. Therefore to develop tolerance in plants to cope with changing environmental condition is need of hour to feed the overgrowing population. In this review, we discussed the application of CRISPR to enhance the crop productivity and develop biotic and abiotic stress-tolerant in potato to face the current changing climatic conditions is also discussed. The review also sheds light on the future prospects of CRISPR/Cas technology for potato breeding, including potential challenges and opportunities. [ABSTRACT FROM AUTHOR]

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated enzyme-CAS holds great promise for treating many uncured human diseases and illnesses by precisely correcting harmful point mutations and disrupting disease-causing genes. The recent Food and Drug Association (FDA) approval of the first CRISPR-based gene therapy for sickle cell anemia marks the beginning of a new era in gene editing. However, delivering CRISPR specifically into diseased cells in vivo is a significant challenge and an area of intense research. The identification of new CRISPR/Cas variants, particularly ultra-compact CAS systems with robust gene editing activities, paves the way for the low-capacity delivery vectors to be used in gene therapies. CRISPR/Cas technology has evolved beyond editing DNA to cover a wide spectrum of functionalities, including RNA targeting, disease diagnosis, transcriptional/epigenetic regulation, chromatin imaging, high-throughput screening, and new disease modeling. CRISPR/Cas can be used to engineer B-cells to produce potent antibodies for more effective vaccines and enhance CAR T-cells for the more precise and efficient targeting of tumor cells. However, CRISPR/Cas technology has challenges, including off-target effects, toxicity, immune responses, and inadequate tissue-specific delivery. Overcoming these challenges necessitates the development of a more effective and specific CRISPR/Cas delivery system. This entails strategically utilizing specific gRNAs in conjunction with robust CRISPR/Cas variants to mitigate off-target effects. This review seeks to delve into the intricacies of the CRISPR/Cas mechanism, explore progress in gene therapies, evaluate gene delivery systems, highlight limitations, outline necessary precautions, and scrutinize the ethical considerations associated with its application. [ABSTRACT FROM AUTHOR]

The study of the functional genome in mice and humans has been instrumental for describing the conserved molecular mechanisms regulating human reproductive biology, and for defining the etiologies of monogenic fertility disorders. Infertility is a reproductive disorder that includes various conditions affecting a couple's ability to achieve a healthy pregnancy. Recent advances in next-generation sequencing and CRISPR/Cas-mediated genome editing technologies have facilitated the identification and characterization of genes and mechanisms that, if affected, lead to infertility. We report established genes that regulate conserved functions in fundamental reproductive processes (e.g., sex determination, gametogenesis, and fertilization). We only cover genes the deletion of which yields comparable fertility phenotypes in both rodents and humans. In the case of newly-discovered genes, we report the studies demonstrating shared cellular and fertility phenotypes resulting from loss-of-function mutations in both species. Finally, we introduce new model systems for the study of human reproductive biology and highlight the importance of studying human consanguineous populations to discover novel monogenic causes of infertility. The rapid and continuous screening and identification of putative genetic defects coupled with an efficient functional characterization in animal models can reveal novel mechanisms of gene function in human reproductive tissues. [ABSTRACT FROM AUTHOR]

At present, there are a variety of different approaches to the targeted regulation of gene expression. However, most approaches are devoted to the activation of gene transcription, and the methods for gene silencing are much fewer in number. In this review, we describe the main systems used for the targeted suppression of gene expression (including RNA interference (RNAi), chimeric transcription factors, chimeric zinc finger proteins, transcription activator-like effectors (TALEs)-based repressors, optogenetic tools, and CRISPR/Cas-based repressors) and their application in eukaryotes—plants and animals. We consider the advantages and disadvantages of each approach, compare their effectiveness, and discuss the peculiarities of their usage in plant and animal organisms. This review will be useful for researchers in the field of gene transcription suppression and will allow them to choose the optimal method for suppressing the expression of the gene of interest depending on the research object. [ABSTRACT FROM AUTHOR]

Gene editing therapies are designed to minimise off-target editing. However, it is not widespread practice for common polymorphisms to be considered when identifying potential off-target sites in silico. Nevertheless, genetic variants should be included as they have the potential to alter existing, or to generate new, off-target sites. To facilitate the consideration of common polymorphisms when designing targeted gene therapies we developed PopOff, a web-based tool that integrates minor allele frequencies from the gnomAD variant database into an off-target analysis. We used PopOff to analyse predicted off-target loci from guide RNAs used in four clinical trials and thirty-four research publications. From an analysis of sixty guides, we identified that approximately 20% of off-target loci overlap with a common polymorphism. Of these sites, 6.93% contained variants that reduce the level of mismatch between the off-target locus and guide, and therefore may increase off-target cleavage. In addition, we identified that 0.34% of common polymorphisms generated novel PAM sites, resulting in off-target loci that standard workflows would miss. Our findings demonstrate that common polymorphisms should be considered when designing guides to maximise the safety of CRISPR-based gene therapies. However, this may be problematic in populations where the breadth of genetic diversity remains uncharacterised. [ABSTRACT FROM AUTHOR]

Summary: Sphingolipids are widespread, abundant, and essential lipids in plants and in other eukaryotes. Glycosyl inositol phosphorylceramides (GIPCs) are the most abundant class of plant sphingolipids, and are enriched in the plasma membrane of plant cells. They have been difficult to study due to lethal or pleiotropic mutant phenotypes.To overcome this, we developed a CRISPR/Cas9‐based method for generating multiple and varied knockdown and knockout populations of mutants in a given gene of interest in the model moss Physcomitrium patens. This system is uniquely convenient due to the predominantly haploid state of the Physcomitrium life cycle, and totipotency of Physcomitrium protoplasts used for transformation.We used this approach to target the INOSITOL PHOSPHORYLCERAMIDE SYNTHASE (IPCS) gene family, which catalyzes the first, committed step in the synthesis of GIPCs. We isolated knockout single mutants and knockdown higher‐order mutants showing a spectrum of deficiencies in GIPC content. Remarkably, we also identified two mutant alleles accumulating inositol phosphorylceramides, the direct products of IPCS activity, and provide our best explanation for this unexpected phenotype.Our approach is broadly applicable for studying essential genes and gene families, and for obtaining unusual lesions within a gene of interest. [ABSTRACT FROM AUTHOR]

Biological nanoparticles (NPs), such as extracellular vesicles (EVs), exosome-mimetic nanovesicles (EMNVs) and nanoghosts (NGs), are perspective non-viral delivery vehicles for all types of therapeutic cargo. Biological NPs are renowned for their exceptional biocompatibility and safety, alongside their ease of functionalization, but a significant challenge arises when attempting to load therapeutic payloads, such as nucleic acids (NAs). One effective strategy involves fusing biological NPs with liposomes loaded with NAs, resulting in hybrid carriers that offer the benefits of both biological NPs and the capacity for high cargo loads. Despite their unique parameters, one of the major issues of virtually any nanoformulation is the ability to escape degradation in the compartment of endosomes and lysosomes which determines the overall efficiency of nanotherapeutics. In this study, we fabricated all major types of biological and hybrid NPs and studied their response to the acidic environment observed in the endolysosomal compartment. In this study, we show that EMNVs display increased protonation and swelling relative to EVs and NGs in an acidic environment. Furthermore, the hybrid NPs exhibit an even greater response compared to EMNVs. Short-term incubation of EMNVs in acidic pH corresponding to late endosomes and lysosomes again induces protonation and swelling, whereas hybrid NPs are ruptured, resulting in the decline in their quantities. Our findings demonstrate that in an acidic environment, there is enhanced rupture and release of vesicular cargo observed in hybrid EMNVs that are fused with liposomes compared to EMNVs alone. This was confirmed through PAGE electrophoresis analysis of mCherry protein loaded into nanoparticles. In vitro analysis of NPs colocalization with lysosomes in HepG2 cells demonstrated that EMNVs mostly avoid the endolysosomal compartment, whereas hybrid NPs escape it over time. To conclude, (1) hybrid biological NPs fused with liposomes appear more efficient in the endolysosomal escape via the mechanism of proton sponge-associated scavenging of protons by NPs, influx of counterions and water, and rupture of endo/lysosomes, but (2) EMNVs are much more efficient than hybrid NPs in actually avoiding the endolysosomal compartment in human cells. These results reveal biochemical differences across four major types of biological and hybrid NPs and indicate that EMNVs are more efficient in escaping or avoiding the endolysosomal compartment. [ABSTRACT FROM AUTHOR]

Hordeum vulgare genes NUD (HvNUD) and WIN1 (HvWIN1) play a regulatory role in cuticle organization. Because the cuticle is a key evolutionary acquisition of plants for protection against environmental factors, a knockout (KO) of each gene may alter their ability to adapt to unfavorable conditions. A potential pleiotropic effect of HvNUD or HvWIN1 gene mutations can be assessed under salt stress. Initial developmental stages are the most sensitive in living organisms; therefore, we evaluated salt tolerance of nud KO and win1 KO barley lines at the seedling stage. Air-dried barley grains of the KO lines and of a wild-type (WT) line were germinated in NaCl solutions (50, 100, or 150 mM). Over 30 physiological and morphological parameters of seedlings were assessed. Potential pleiotropic effects of the HvNUD gene KO under salt stress included the stimulation of root growth (which was lower under control conditions) and root necrosis. The pleiotropic effects of the HvWIN1 gene KO under the stressful conditions manifested themselves as maintenance of longer root length as compared to the other lines; stable variation of most of morphological parameters; lack of correlation between root lengths before and after exposure to NaCl solutions, as well as between shoot lengths; and the appearance of twins. Salt tolerance of the analyzed barley lines could be ranked as follows: nud KO > win1 KO ≈ WT, where nud KO lines were the most salt-tolerant. A comparison of effects of salinity and ionizing radiation on nud KO and win1 KO barley lines indicated differences in tolerance of the lines to these stressors. [ABSTRACT FROM AUTHOR]

The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. Machado–Joseph Disease (MJD) or spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant form, caused by the expansion of CAG repeats within the ataxin-3 (ATXN3) gene. This mutation results in the expression of an abnormal protein containing long polyglutamine (polyQ) stretches that confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. As a result of the neurodegenerative process, SCA3 patients are severely disabled and die prematurely. Several screening approaches, e.g., druggable genome-wide and drug library screenings have been performed, focussing on the reduction in stably overexpressed ATXN3(polyQ) protein and improvement in the resultant toxicity. Transgenic overexpression models of toxic ATXN3, however, missed potential modulators of endogenous ATXN3 regulation. In another approach to identify modifiers of endogenous ATXN3 expression using a CRISPR/Cas9-modified SK-N-SH wild-type cell line with a GFP-T2A-luciferase (LUC) cassette under the control of the endogenous ATXN3 promotor, four statins were identified as potential activators of expression. We here provide an overview of the high throughput screening approaches yet performed to find compounds or genomic modifiers of ATXN3(polyQ) toxicity in different SCA3 model organisms and cell lines to ameliorate and halt SCA3 progression in patients. Furthermore, the putative role of cholesterol in neurodegenerative diseases (NDDs) in general and SCA3 in particular is discussed. [ABSTRACT FROM AUTHOR]

Soybean is one of the richest and cheapest proteins and vegetable oil sources. It is adapted to grow in a varied environment; however, yield loss occurs due to multiple abiotic stresses. Abiotic stresses negatively impact plant growth and development, damaging the crop and decreasing productivity. The last two decades have focused tremendously on improving soybean productivity by dissecting physiological and molecular mechanisms for developing abiotic stress-tolerant varieties. Here, we present a review with a comprehensive outlook on the biotechnological approaches to explore the pathways involved in abiotic stress tolerance in soybean. The review focuses on summarizing transgenic and RNA interference-based strategies as well as genome editing tools to validate the function of abiotic stress-associated genes in soybean. We have also highlighted the significant challenges faced in increasing soybean yield against climatic changes using diverse techniques. [ABSTRACT FROM AUTHOR]

BACKGROUND: Verticillium dahliae is a soil‐borne pathogenic fungus that causes Verticillium wilt disease on more than 400 plant species worldwide. Because of its broad host range and its ability to survive long term in the soil, there are few effective control measures for V. dahliae once it has become established. Accurate, sensitive, and rapid detection of V. dahliae is crucial for limiting pathogen entry into new regional environments and early management of Verticillium wilt. RESULTS: In this study, we developed a method to detect V. dahliae based on recombinase polymerase amplification (RPA) and CRISPR/Cas technology and used fluorescence and lateral flow test strips to monitor the outcomes. Through the establishment and optimization of RPA–CRISPR/Cas13a detection, the sensitivity of the fluorescence method was 1 am for genomic DNA (gDNA) within 20 min, whereas the sensitivity of the lateral flow strip method was 100 am for gDNA in 30 min. The field applicability of RPA–CRISPR/Cas13a was also validated by the detection of V. dahliae on smoke trees (Cotinus coggygria) in Xiangshan Park, Beijing, China. Finally, diplex detection for defoliating and nondefoliating pathotypes of V. dahliae was established by combining CRISPR–Cas12a/Cas13a with specific target genes. CONCLUSION: Taken together, this study achieved rapid, sensitive, and accurate detection of V. dahliae and the differentiation of defoliating and nondefoliating pathotypes and provides potential for field‐deployable diagnostic tools for rapid and ultrasensitive detection. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Tumor‐derived exosomes are crucial for early non‐invasive and accurate tumor diagnosis in clinical diagnostics. The development of highly sensitive, simple, and intuitive exosome assays has sparked a research upsurge in clinical diagnostics. Here, we develop a bio‐responsive intelligent DNA hydrogel loaded with CRISPR/Cas12a for universal and ultrasensitive detection of the exosomes. The aptamer serves as the target response unit and switch, competitively disintegrating the region of the DNA linkers and then Cas12a/crRNA was released and activated, resulting in a high fluorescent intensity for exosome detection at the detection limit of 119 particles/μL. Moreover, a constructed colorimetric tube is made by loading a colorimetric filter membrane on the tube lid and intelligent DNA hydrogel on the tube bottom, which enables one‐pot portable colorimetric detection. Without the need for laboratory instruments and professionals, this strategy allows for naked eye detection with limit of detection as low as 104 particles/μL, and shows great applicability in distinguishing between healthy individuals, pretreatment patients, and post‐treatment patients after obtaining a testable analyte. In this study, an ultrasensitive detection platform for exosomes that enables one‐step sensing and dual signal output was introduced. The findings here suggest that this platform is a promising tool for the application of liquid biopsy based on exosomes in clinical diagnosis. [ABSTRACT FROM AUTHOR]

Millets, the sixth most-grown group of crops in the drylands, support the livelihood of many small-holder farmers in the region. Being one of the most nutritious groups of crops, their production has been increasing since the last decade to meet the demands of the world's ever-increasing population. Since its discovery, CRISPR/Cas-mediated gene editing technology has revolutionized trait improvement in numerous crops by enabling targeted insertions and deletions at specific gene sequences. With advancements like base editing and prime editing, which offer precise modifications at the nucleotide level, this technology holds great promise for enhancing millets by targeting genes responsible for key traits. The updated sequence information in the public domain makes it possible to modify certain genic regions using the CRISPR/Cas-mediated gene editing technology to develop millet crops with improved agronomical properties. The review explores each component of the editing toolbox in millets, including the gRNA designing tools, types of Cas nucleases, and promoters to be considered for enhanced and efficient gene editing in millets. We have discussed fundamental information available to successfully employ CRISPR/Cas-mediated gene editing in millets, such as the availability of genomic information and plant transformation methods. Finally, we have highlighted the limitations of employing this novel technology in millet crops by providing future directions and immediate candidate genes that could be targeted to improve various traits in millet crops. [ABSTRACT FROM AUTHOR]

Gene editing and therapy holds immense promise in addressing cardiovascular disorders by enabling precise modifications to the genetic groundworks of these conditions. This technology offers a targeted approach to correct or mitigate not only the genetic mutations that are responsible for inherited cardiovascular diseases, such as hypertrophic cardiomyopathy or familial hypercholesterolemia (FH), but also in controlling and regulating the genes in conditions that are age-related or that arise due to unnecessary gene activity, which often lack effective treatments. By editing the relevant genes, we aim to reduce disease severity, improve heart function, prevent future episodes and potentially improve the health span. Recent trials involving gene therapy and gene editing in cardiovascular disorders have revolutionized treatment strategies, offering hope for patients with genetic predispositions to heart and vessel-related ailments and advancing the pursuit for more personalized and effective therapies. In this review, we have consolidated the genetic mutations causing cardiovascular diseases (CVDs) followed by latest advancements in the gene editing technologies and their therapeutic implications along with involved ethical challenges and risk factors. [ABSTRACT FROM AUTHOR]