Genome Editing Research Articles

Real-Time Genomic Analytics in Clinical Practice: A Framework for High-Throughput Data Processing and Decision Support

Recent advances in genomic sequencing technologies have generated unprecedented volumes of clinical genomic data, necessitating robust real-time analytics solutions for immediate clinical decision support. This article presents a comprehensive framework for implementing real-time genomic data processing in clinical settings, addressing the challenges of high-throughput data management while maintaining patient privacy and data security. The article examines the integration of distributed computing frameworks and stream processing technologies to facilitate rapid analysis of genomic data alongside clinical phenotypic information. The article reveals that modern healthcare informatics platforms can effectively manage multi-modal datasets through optimized data pipelines, enabling faster diagnostic processes and improved patient outcomes. The article demonstrates how real-time analytics can enhance clinical decision-making through immediate variant calling and interpretation while supporting larger population-scale genomic studies. The article discusses solutions to critical challenges in data quality management, privacy preservation, and computational resource optimization. The findings suggest that real-time genomic analytics can significantly improve diagnostic speed and accuracy while advancing preventative healthcare strategies through better identification of genetic risk factors. This article contributes to the growing field of precision medicine by providing a scalable approach to managing and analyzing genomic data in time-critical clinical environments.

Approaches to Incorporation of Preferences into Health Economic Models of Genomic Medicine: A Critical Interpretive Synthesis and Conceptual Framework.

Genomic medicine has features that make it preference sensitive and amenable to model-based health economic evaluation. Preferences of patients, caregivers, and clinicians related to the uptake and delivery of genomic medicine technologies and services that are not captured in health state utility weights can affect the intervention's cost-effectiveness and budget impact. However, there is currently no established or agreed-on approach for integrating preference information into economic evaluations. The objective of this study was to explore approaches for incorporating preferences into model-based economic evaluations of genomic medicine and to develop a conceptual framework to consider preferences in health economic models. We conducted a critical interpretive synthesis of published literature guided by the following question: how have preferences been incorporated into model-based economic evaluations of genomic medicine interventions? We integrated findings from the literature and expert opinion to develop a conceptual framework of ways in which preferences influence economic value in the context of genomic medicine. Our synthesis included 14 articles. Revealed and stated preference data were used to estimate choice probabilities and to value outcomes. Our conceptual framework situates preference data in the context of health system, patient, clinician, and family characteristics. Preference data were sourced from clinicians, patients and families impacted by a condition or intervention, and the general public. Evaluations employed various types of models, including discrete event simulation, microsimulation, Markov, and decision tree models. When evaluating the broad benefits and costs of implementing new interventions, sufficiently accounting for preferences in the form of model inputs and valuation of outcomes in economic evaluations is important to avoid biased implementation decisions. Incorporation of preference data may improve alignment between predicted and real-world uptake and more accurately estimate welfare impacts, and this study provides critical insights to support researchers who seek to incorporate preference information into model-based health economic evaluations.

CRISPR/Cas9-mediated genomic editing of Brachytic2 creates semi-dwarf mutant alleles for tailored maize breeding.

CRISPR/Cas9-mediated genomic editing of Brachytic2 creates semi-dwarf mutant alleles for tailored maize breeding.

Prevalent integration of genomic repetitive and regulatory elements and donor sequences at CRISPR-Cas9-induced breaks

CRISPR-Cas9 genome editing has been extensively applied in both academia and clinical settings, but its genotoxic risks, including large insertions (LgIns), remain poorly studied due to methodological limitations. This study presents the first detailed report of unintended LgIns consistently induced by different Cas9 editing regimes using various types of donors across multiple gene loci. Among these insertions, retrotransposable elements (REs) and host genomic coding and regulatory sequences are prevalent. RE frequencies and 3D genome organization analysis suggest LgIns originate from randomly acquired genomic fragments by DNA repair mechanisms. Additionally, significant unintended full-length and concatemeric double-stranded DNA (dsDNA) donor integrations occur when donor DNA is present. We further demonstrate that phosphorylated dsDNA donors consistently reduce large insertions and deletions by almost two-fold without compromising homology-directed repair (HDR) efficiency. Taken together, our study addresses a ubiquitous and overlooked risk of unintended LgIns in Cas9 editing, contributing valuable insights for the safe use of Cas9 editing tools.

The generation and evaluation of TKO/hCD55/hTM/hEPCR gene-modified pigs for clinical organ xenotransplantation.

Genetically edited pigs, modified using CRISPR-Cas9 technology, hold promise as potential sources for xenotransplantation. However, the optimal combination of genetic modifications and their expression levels for initial clinical trials remains unclear. This study investigates the generation of TKO/hCD55/hTM/hEPCR (6GE) pigs and evaluates their compatibility with human immune and coagulation systems. The 6GE pigs were generated through iterative genome editing and F1 generation breeding. Genotyping, flow cytometry, and immunohistochemistry confirmed the knockout of GGTA1, CMAH, and B4GALNT2. Expression levels of human genes (hCD55, hTM, hEPCR) were quantified. In vitro assays using aortic endothelial cells (pAECs) from 6GE pigs assessed human serum IgM and IgG binding, complement cytotoxicity, and thrombin-antithrombin (TAT) complex levels. Blood from gene-edited pigs was used for pathophysiological analysis. Complete knockout of GGTA1, CMAH, and B4GALNT2 was confirmed in 6GE pigs. The expression of hCD55 and hTM was approximately seven and thirteen times higher than in humans, respectively, while hEPCR levels were comparable to those in humans. In vitro, 6GE pAECs showed significantly reduced binding of human IgM and IgG compared to wild-type pAECs (IgG p<0.01, IgM p<0.0001). Similar to TKO/hCD55 pAECs, 6GE pAECs exhibited a substantial reduction in complement-mediated cytotoxicity (p<0.001) compared to TKO pAECs. Co-expression of hTM and hEPCR in 6GE pigs led to a significant decrease in thrombin-antithrombin (TAT) complex levels in co-culture with human whole blood, compared to WT (p<0.0001), TKO (p<0.01), and TKO/hCD55/hTM pigs (p<0.05). Pathophysiological analysis demonstrated excellent compatibility of 6GE pig kidneys and livers with human immune and coagulation systems. However, 6GE pigs showed increased susceptibility to infection compared to other gene-edited pigs, while TKO/hCD55 pigs were considered safe when they were all bred in a general environment. Highly expressing hCD55, along with the co-expression of hEPCR and hTM genes, is expected to effectively reduce human complement cytotoxicity and enhance anticoagulant efficacy in genetically modified pigs. The 6GE pigs exhibited robust compatibility with human physiological and immune systems, fulfilling the criteria for clinical trials. Furthermore, it is imperative to rear donor pigs in pathogen-free (DPF) facilities to mitigate infection risks and prevent the transmission of porcine pathogens to humans.

Comparing three emerging industrial cell factories: Pseudomonas putida KT2440, Halomonas bluephagenesis TD01, and Zymomonas mobilis ZM4.

Nonmodel microbes with unique advantages are emerging as industrial platforms, driven by advances in genetic engineering and omics technologies. Notable examples include the versatile soil bacterium Pseudomonas putida KT2440, the halophilic Halomonas bluephagenesis TD01, and the ethanologenic Zymomonas mobilis ZM4. While all three primarily use the Entner-Doudoroff pathway for glucose metabolism, they differ in various metabolic pathways and product synthesis. This review summarizes and compares their central carbon metabolism, advancements in genome engineering tools, and progress in scaling industrial applications from lab scale, to pilot scale, to full-scale commercial production. Understanding their similarities and differences informs future research on optimizing industrial applications and may guide the development of new microbial hosts.

Zymomonas mobilis: bringing an ancient human tool into the genomic era.

Zymomonas mobilis is an ethanologenic bacterium that has been used for over 1500 years to produce alcoholic beverages. Recently, this microbe has become a top candidate for biofuel production due to its efficient metabolism. Z. mobilis is being developed to utilize lignocellulosic biomass as a feedstock and synthesize a range of valuable chemicals and fuels. Genetic and metabolic engineering strategies are crucial to reach these goals. Recent advances include genome engineering, CRISPR editing, and CRISPRi knockdown of genes. Metabolic engineering has enabled redirection of carbon from the natural product ethanol to chemicals such as 2,3-butanediol and polyhydroxybutyrate. The approaches summarized here will streamline the development of Z. mobilis as an industrial chassis for sustainable liquid fuels and chemicals.

Application of Genomic Selection in Beef Cattle Disease Prevention.

Genomic applications in beef cattle disease prevention have gained traction in recent years, offering new strategies for improving herd health and reducing economic losses in the livestock industry. Advances in genomics, including identification of genetic markers linked to disease resistance, provide powerful tools for early detection, selection, and management of cattle resistant to infectious diseases. By incorporating genomic technologies such as whole-genome sequencing, genotyping, and transcriptomics, researchers can identify specific genetic variants associated with resistance to pathogens like bovine respiratory disease and Johne's disease. These genomic insights allow for more accurate breeding programs aimed at enhancing disease resistance and overall herd resilience. Genomic selection, in particular, enables identification of individuals with superior genetic traits for immune function, reducing the need for antibiotic treatments and improving animal welfare. Moreover, precision medicine, powered by genomic data, supports development of tailored health management strategies, including targeted vaccination plans and antimicrobial stewardship. Incorporation of genomic tools in beef cattle management also offers the potential for early disease detection, facilitating proactive interventions that reduce the spread of infections. Despite challenges like cost, data interpretation and integration into current management systems, the potential advantages of genomic applications in disease prevention are substantial. As these technologies advance, they are anticipated to have crucial roles in improving sustainability (by enhancing herd performance), profitability (by improving overall herd longevity), and biosecurity (by decreasing the likelihood of disease outbreaks) of beef cattle production systems worldwide.

CRISPR-Cas12: A Versatile Tool for Genome Editing and Beyond

CRISPR-Cas systems are emerging gene editing toolkits that have been used for the manipulation of DNA and RNA, among which CRISPR-Cas12 demonstrates superior properties in terms of target DNA engineering in vivo compared to CRISPR-Cas9 as well as nucleic acid detection compared to CRISPR-Cas13. A comprehensive understanding of the mechanisms and emerging applications of CRISPR-Cas12-based technologies is critical for maximizing their benefits across various fields, including basic science, medicine, and food safety. In this study, we review the molecular features of both natural and engineered CRISPR-Cas12 compared to CRISPR-Cas9 and CRISPR-Cas13, and we provide an overview of established CRISPR-Cas12-based genome editing and diagnostic platforms. Furthermore, we discuss the advantages, challenges, and prospects for future directions of CRISPR-Cas12 in gene editing and nucleic acid detection.

Elucidating role of long non-coding RNAs of Tamarindus indica Linn. in post-transcriptional gene regulation

Tamarindus indica Linn., commonly known as tamarind, is a rich source of carbohydrates, proteins, lipids, fatty acids, vitamins, minerals and bioactive compounds. It is well established that long non-coding RNAs (lncRNAs) plays an important role in transcriptional, post-transcriptional and epigenetic regulation. Despite the availability of tamarind genome information, a handful of studies have been done on its non-coding genome, especially lncRNAs. In this study, we have computationally predicted lncRNAs from the coding DNA sequences of T. indica and analysed the sequences. We experimentally validated seven randomly chosen lncRNAs by performing quantitative Real Time Polymerase Chain Reaction (qRT-PCR). 320 lncRNAs have been predicted and sequence analysis of these predicted, lncRNAs reveals the presence of different motifs and tandem repeats. Along with the experimental validation of 7 randomly chosen lncRNAs, functional analysis of the predicted lncRNAs and their targets elucidated their roles in various biological pathways. We believe prediction and validation of the lncRNAs along with their interaction with mRNAs will enhance our knowledge about the non-coding genome of tamarind and their involvement in post transcriptional gene regulation, medicinal properties, metabolic engineering, stress tolerance and genome editing.

Groundbreaking Technologies and the Biocontrol of Fungal Vascular Plant Pathogens.

This review delves into innovative technologies to improve the control of vascular fungal plant pathogens. It also briefly summarizes traditional biocontrol approaches to manage them, addressing their limitations and emphasizing the need to develop more sustainable and precise solutions. Powerful tools such as next-generation sequencing, meta-omics, and microbiome engineering allow for the targeted manipulation of microbial communities to enhance pathogen suppression. Microbiome-based approaches include the design of synthetic microbial consortia and the transplant of entire or customized soil/plant microbiomes, potentially offering more resilient and adaptable biocontrol strategies. Nanotechnology has also advanced significantly, providing methods for the targeted delivery of biological control agents (BCAs) or compounds derived from them through different nanoparticles (NPs), including bacteriogenic, mycogenic, phytogenic, phycogenic, and debris-derived ones acting as carriers. The use of biodegradable polymeric and non-polymeric eco-friendly NPs, which enable the controlled release of antifungal agents while minimizing environmental impact, is also explored. Furthermore, artificial intelligence and machine learning can revolutionize crop protection through early disease detection, the prediction of disease outbreaks, and precision in BCA treatments. Other technologies such as genome editing, RNA interference (RNAi), and functional peptides can enhance BCA efficacy against pathogenic fungi. Altogether, these technologies provide a comprehensive framework for sustainable and precise management of fungal vascular diseases, redefining pathogen biocontrol in modern agriculture.

Unlocking the potential of CRISPR-Cas9 for cystic fibrosis: A systematic literature review.

CRISPR-Cas9 technology has revolutionized genetic engineering, offering precise and efficient genome editing capabilities. This review explores the application of CRISPR-Cas9 for cystic fibrosis (CF), particularly targeting mutations in the CFTR gene. CF is a multiorgan disease primarily affecting the lungs, gastrointestinal system (e.g., CF-related diabetes (CFRD), CF-associated liver disease (CFLD)), bones (CF-bone disease), and the reproductive system. CF, a genetic disorder characterized by defective ion transport leading to thick mucus accumulation, is often caused by mutations like ΔF508 in the CFTR gene. This review employs a systematic methodology, incorporating an extensive literature search across multiple academic databases, including PubMed, Web of Science, and ScienceDirect, to identify 40 high-quality studies focused on CRISPR-Cas9 applications for CFTR gene editing. The data collection process involved predefined inclusion criteria targeting experimental approaches, gene-editing outcomes, delivery methods, and verification techniques. Data analysis synthesized findings on editing efficiency, off-target effects, and delivery system optimization to present a comprehensive overview of the field. The review highlights the historical development of CRISPR-Cas9, its mechanism, and its transformative role in genetic engineering and medicine. A detailed examination of CRISPR-Cas9's application in CFTR gene correction emphasizes the potential for therapeutic interventions while addressing challenges such as off-target effects, delivery efficiency, and ethical considerations. Future directions include optimizing delivery systems, integrating advanced editing tools like prime and base editing, and expanding personalized medicine approaches to improve treatment outcomes. By systematically analyzing the current landscape, this review provides a foundation for advancing CRISPR-Cas9 technologies for cystic fibrosis treatment and related disorders.

Advancing Insights into Pediatric Macular Diseases: A Comprehensive Review.

Pediatric macular disorders are a diverse group of inherited retinal diseases characterized by central vision loss due to dysfunction and degeneration of the macula, the region of the retina responsible for high-acuity vision. Common disorders in this category include Stargardt disease, Best vitelliform macular dystrophy, and X-linked retinoschisis. These conditions often manifest during childhood or adolescence, with symptoms such as progressive central vision loss, photophobia, and difficulty with fine visual tasks. Underlying mechanisms involve genetic mutations that disrupt photoreceptor and retinal pigment epithelium function, accumulating toxic byproducts, impaired ion channel activity, or structural degeneration. Advances in imaging modalities like optical coherence tomography and fundus autofluorescence have improved diagnostic accuracy and disease monitoring. Emerging therapies are transforming the treatment landscape. Gene therapy and genome editing hold promise for addressing the genetic basis of these disorders, while stem cell-based approaches and pharmacological interventions aim to restore retinal function and mitigate damage. Personalized medicine, driven by genomic sequencing, offers the potential for tailored interventions. Despite current challenges, ongoing research into molecular mechanisms, advanced imaging, and innovative therapies provides hope for improving outcomes and quality of life in children with macular disorders.

Treating genetic blood disorders in the era of CRISPR-mediated genome editing.

In the setting of monogenic disease, advances made in genome editing technologies can, in principle, be deployed as a therapeutic strategy to precisely correct a specific gene mutation in an affected cell type and restore functionality. Using the β-hemoglobinopathies and hemophilia as exemplars, we review recent experimental breakthroughs utilizing CRISPR-derived genome editing technology that have translated to significant improvements in the management of inherited hematologic disorders. Yet there are also challenges facing the use of CRISPR mediated genome editing in these patients and we discuss possible ways to obviate those issues for furtherance of clinical benefit.

Short- and long-range roles of UNC-6/Netrin in dorsal-ventral axon guidance in vivo in Caenorhabditis elegans.

Recent studies in vertebrates and Caenorhabditis elegans have reshaped models of how the axon guidance cue UNC-6/Netrin functions in dorsal-ventral axon guidance, which was traditionally thought to form a ventral-to-dorsal concentration gradient that was actively sensed by growing axons. In the vertebrate spinal cord, floorplate Netrin1 was shown to be largely dispensable for ventral commissural growth. Rather, short range interactions with Netrin1 on the ventricular zone radial glial stem cells was shown to guide ventral commissural axon growth. In C. elegans, analysis of dorsally-migrating growth cones during outgrowth has shown that growth cone polarity of filopodial extension is separable from the extent of growth cone protrusion. Growth cones are first polarized by UNC-6/Netrin, and subsequent regulation of protrusion by UNC-6/Netrin is based on this earlier-established polarity (the Polarity/Protrusion model). In both cases, short-range or even haptotactic mechanisms are invoked: in vertebrate spinal cord, interactions of growth cones with radial glia expressing Netrin-1; and in C. elegans, a potential close-range interaction that polarizes the growth cone. To explore potential short-range and long-range functions of UNC-6/Netrin, a potentially membrane-anchored transmembrane UNC-6 (UNC-6(TM)) was generated by genome editing. unc-6(tm) was hypomorphic for dorsal VD/DD axon pathfinding, indicating that it retained some unc-6 function. Polarity of VD growth cone filopodial protrusion was initially established in unc-6(tm), but was lost as the growth cones migrated away from the unc-6(tm) source in the ventral nerve cord. In contrast, ventral guidance of the AVM and PVM axons was equally severe in unc-6(tm) and unc-6(null). Together, these results suggest that unc-6(tm) retains short-range functions but lacks long-range functions due to reduced secreted UNC-6. Ectopic unc-6(+) expression from non-ventral sources did not dramatically perturb dorsal VD growth cone polarity or axon outgrowth, suggesting that ectopic UNC-6 cannot redirect polarity once it is established in the VD/DD neurons. This is not what would be expected of a growth cone dynamically reading a gradient of UNC-6, but is consistent with the Polarity/protrusion model of growth cone guidance away from UNC-6/Netrin.

Improving Crop Tolerance to Abiotic Stress for Sustainable Agriculture: Progress in Manipulating Ascorbic Acid Metabolism via Genome Editing

Improving Crop Tolerance to Abiotic Stress for Sustainable Agriculture: Progress in Manipulating Ascorbic Acid Metabolism via Genome Editing

CRISPR-Cas9 in Cardiovascular Medicine: Unlocking New Potential for Treatment.

Cardiovascular diseases (CVDs) remain a significant global health challenge, with many current treatments addressing symptoms rather than the genetic roots of these conditions. The advent of CRISPR-Cas9 technology has revolutionized genome editing, offering a transformative approach to targeting disease-causing mutations directly. This article examines the potential of CRISPR-Cas9 in the treatment of various CVDs, including atherosclerosis, arrhythmias, cardiomyopathies, hypertension, and Duchenne muscular dystrophy (DMD). The technology's ability to correct single-gene mutations with high precision and efficiency positions it as a groundbreaking tool in cardiovascular therapy. Recent developments have extended the capabilities of CRISPR-Cas9 to include mitochondrial genome editing, a critical advancement for addressing mitochondrial dysfunctions often linked to cardiovascular disorders. Despite its promise, significant challenges remain, including off-target effects, ethical concerns, and limitations in delivery methods, which hinder its translation into clinical practice. This article also explores the ethical and regulatory considerations surrounding gene editing technologies, emphasizing the implications of somatic versus germline modifications. Future research efforts should aim to enhance the accuracy of CRISPR-Cas9, improve delivery systems for targeted tissues, and ensure the safety and efficacy of treatments in the long term. Overcoming these obstacles could enable CRISPR-Cas9 to not only treat but also potentially cure genetically driven cardiovascular diseases, heralding a new era in precision medicine for cardiovascular health.

Drug Repurposing: A Conduit to Unravelling Metabolic Reprogramming for Cancer Treatment.

Metabolic reprogramming is a hallmark of cancer. Distinct and unusual metabolic aberrations occur during tumor development that lead to the growth and development of tumors. Oncogenic signaling pathways eventually converge to regulate three major metabolic pathways in tumor cells i.e., glucose, lipid, and amino acid metabolism. Therefore, identifying and targeting the metabolic nodes of cancer cells can be a promising intervention and therapeutic strategy for patients with malignancies. The long road of new drug discovery for cancer therapy has necessitated relooking alternative strategies such as drug repurposing. Advanced genomic and proteomic technologies for the assessment of cancer-specific biological pathways have led to the discovery of new drug targets, which provide excellent opportunities for drug repurposing. The development of effective, safe, cheaper, and readily available anticancer agents is the need of the hour, and drug repurposing has the potential to break the current drug shortage bottleneck. This review will accordingly cover various metabolic pathways that are aberrant in cancer, and strategies for targeting metabolic reprogramming by using repurposed drugs.

Large donor CRISPR for whole-CDS replacement of cell adhesion molecule LRRTM2.

The cell adhesion molecule Leucine-Rich Repeat Transmembrane neuronal protein 2 (LRRTM2) is crucial for synapse development and function. However, our understanding of its endogenous trafficking has been limited due to difficulties in manipulating its coding sequence (CDS) using standard genome editing techniques. Instead, we replaced the entire LRRTM2 CDS by adapting a two-guide CRISPR knock-in method, enabling complete control of LRRTM2. In primary rat hippocampal cultures dissociated from embryos of both sexes, N-terminally tagged, endogenous LRRTM2 was found in 80% of synapses, and synaptic LRRTM2 content correlated with PSD-95 and AMPAR levels. LRRTM2 was also enriched with AMPARs outside synapses, demonstrating the sensitivity of this method to detect relevant new biology. Finally, we leveraged total genomic control to increase the synaptic levels of LRRTM2 via simultaneous mutation of its C-terminal domain, which did not correspondingly increase AMPAR enrichment. The coding region of thousands of genes span lengths suitable for whole-CDS replacement, suggesting this simple approach will enable straightforward structure-function analysis in neurons.Significance Statement Synaptic cell adhesion molecules are transmembrane proteins vital for neurotransmission, and their genes are frequently linked to disease. Mechanistic studies are challenging, however, because overexpression alters their protein trafficking and perturbs neuronal function, yet traditional gene-editing techniques typically permit manipulation at just single sites. Here, we replaced the entire coding sequence (CDS) of the critical adhesion molecule Leucine-Rich Repeat Transmembrane protein 2 (LRRTM2) with a freely editable custom donor sequence, and made the approach even more versatile through introduction of knock-in-dependent cell markers. Using whole-CDS replacement, we discover new aspects of LRRTM2 subcellular distribution and test its role in regulating synaptic proteins. The approach should be suited to structure-function analysis of many other neuronal proteins in their endogenous genetic locus.

Novel Breeding Techniques and Strategies for Enhancing Greenhouse Vegetable Product Quality

With its controlled environment, protected cultivation is advantageous and effective for breeding programs. This distinct setting also guarantees that fresh vegetables meet high quality standards. The controlled environment allows for precise monitoring and tuning of breeding efforts, a critical factor in continuously improving the quality of fresh vegetable production. Classical breeding strategies include hybridization, pedigree selection, backcrossing, recombination, and marker-assisted breeding. However, advanced techniques like phenomics and genome editing are revolutionizing the field. These methods accelerate phenotyping and aid in identifying traits and genetic variants linked to quality characteristics. Modern biotechnological tools, specifically genetic engineering and gene editing methods like CRISPR/Cas, have enhanced a wide array of traits in numerous vegetable species. These technological advancements have the potential to effectively address challenges associated with stress resistance, product quality, and shelf-life, thereby presenting promising prospects for the advancement of agriculture. The protracted process of developing new vegetable cultivars with reduced physiological issues through contemporary techniques is an enduring endeavor.