Gene Editing Research Articles

Utilizing CRISPR-based genetic modification for precise control of seed dormancy: progress, obstacles, and potential directions.

Seed dormancy, a complex trait that is influenced by both nuclear and cytoplasmic factors, poses a significant challenge to agricultural productivity. Conventional dormancy-breaking techniques, including mechanical, physiological, and chemical methods, often yield inconsistent results, impair seed quality, and lack precision. This has necessitated exploration of more targeted and efficient approaches. CRISPR-based gene editing has emerged as a promising tool for the precise regulation of seed dormancy without compromising seed viability or sustainability. Although CRISPR has been successfully applied to modify genes that govern physiological traits in various crops, its use in dormancy regulation remains in the early stages. This review examines recent advancements in CRISPR-based approaches for modulating seed dormancy and discusses key gene targets, modification techniques, and the resulting effects. We also consider the future potential of CRISPR to enhance dormancy control across diverse crop species.

Therapeutic Reprogramming toward Regenerative Medicine.

Therapeutic reprogramming represents a transformative paradigm in regenerative medicine, developing new approaches in cell therapy, small molecule drugs, biologics, and gene therapy to address unmet medical challenges. This paradigm encompasses the precise modulation of cellular fate and function to either generate safe and functional cells ex vivo for cell-based therapies or to directly reprogram endogenous cells in vivo or in situ for tissue repair and regeneration. Building on the discovery of induced pluripotent stem cells (iPSCs), advancements in chemical modulation and CRISPR-based gene editing have propelled a new iterative medicine paradigm, focusing on developing scalable, standardized cell therapy products from universal starting materials and enabling iterative improvements for more effective therapeutic profiles. Beyond cell-based therapies, non-cell-based therapeutic strategies targeting endogenous cells may offer a less invasive, more convenient, accessible, and cost-effective alternative for treating a broad range of diseases, potentially rejuvenating tissues and extending healthspan.

Manipulation of the brown glume and internode 1 gene leads to alterations in the colouration of lignified tissues, lignin content and pathogen resistance in wheat.

Lignin is a crucial component of the cell wall, providing mechanical support and protection against biotic and abiotic stresses. However, little is known about wheat lignin-related mutants and their roles in pathogen defence. Here, we identified an ethyl methanesulfonate (EMS)-derived Aegilops tauschii mutant named brown glume and internode 1 (bgi1), which exhibits reddish-brown pigmentation in various tissues, including internodes, spikes and glumes. Using map-based cloning and single nucleotide polymorphism (SNP) analysis, we identified AET6Gv20438400 (BGI1) as the leading candidate gene, encoding the TaCAD1 protein. The mutation occurred in the splice acceptor site of the first intron, resulting in a premature stop codon in BGI1. We validated the function of BGI1 using loss-of-function EMS and gene editing knockout mutants, both of which displayed reddish-brown pigmentation in lignified tissues. BGI1 knockout mutants exhibited reduced lignin content and shearing force relative to wild type, while BGI1 overexpression transgenic plants showed increased lignin content and enhanced disease resistance against common root rot and Fusarium crown rot. We confirmed that BGI1 exhibits CAD activity both in vitro and in vivo, playing an important role in lignin biosynthesis. BGI1 was highly expressed in the stem and spike, with its localisation observed in the cytoplasm. Transcriptome analysis revealed the regulatory networks associated with BGI1. Finally, we demonstrated that BGI1 interacts with TaPYL-1D, potentially involved in the abscisic acid signalling pathway. The identification and functional characterisation of BGI1 significantly advance our understanding of CAD proteins in lignin biosynthesis and plant defence against pathogen infection in wheat.

Identification of a central regulator of ginkgolide biosynthesis in Ginkgo biloba that integrates jasmonate and light signaling

Ginkgolides are secondary metabolites unique to Ginkgo biloba with the potential to prevent and treat cardiovascular and cerebrovascular diseases. Although the biosynthetic pathways of ginkgolides have been partly uncovered, the mechanism regulating their biosynthesis is still largely unknown. Here, using multiomic and genetic analyses, we report the identification of a transcription factor, named ETHYLENE RESPONSE FACTOR ASSOCIATED WITH GINKGOLIDE BIOSYNTHESIS (GbEAG), as a critical regulator of ginkgolide biosynthesis. GbEAG is highly expressed in the roots of G. biloba, and its expression is significantly induced by methyl jasmonate (MeJA). Ginkgolide content was significantly increased in roots by overexpressing GbEAG using a "cut-dip-regeneration" system. GbEAG positively regulates ginkgolide biosynthesis by directly binding to the GCC-boxes in the promoter regions of genes involved in the biosynthesis of ginkgolides, such as ISOPENTENYL DIPHOSPHATE ISOMERASE (GbIDI) and CYTOCHROME P450 7005C3 (GbCYP7005C3). GbEAG mediates the jasmonic acid (JA)-activated ginkgolide synthesis through its direct interaction with the JASMONATE ZINC-FINGER INFLORESCENCE MERISTEM DOMAIN 3 (GbJAZ3) repressor. Importantly, we also found that the central light-response regulator ELONGATED HYPOCOTYL 5 (GbHY5) mediates light induction of ginkgolide biosynthesis by binding to the G-box in the GbEAG promoter. Our findings provide mechanistic insights into the coordinated regulation of ginkgolide biosynthesis via JA and light signals, with GbEAG as a central regulator in G. biloba, and shed light on the potential to develop ginkgolide-rich varieties through molecular breeding and gene editing.

A universal method for the purification of C2H2 zinc finger arrays.

Zinc fingers (ZFs) are compact, modular, sequence-specific polynucleotide-binding domains uniquely suited for use as DNA probes and for the targeted delivery of effector domains for purposes such as gene regulation and editing. Despite recent advances in both the design and application of ZF-containing proteins, there is still a lack of a general method for their expression and purification. Here we describe a simple method, involving two chromatographic steps, for the production of homogeneous, functional ZF proteins in high yield (one milligram per liter of bacterial culture), and we demonstrate the generality of this method by applying it to a diverse set of eight C2H2-type ZF proteins. By incorporating a surface-exposed terminal cysteine residue that enables site-specific conjugation with maleimide-activated fluorophores, we confirm the suitability of these probes for in situ labeling of specific DNA sequences in human cells.

Gene mutation estimations via mutual information and Ewens sampling based CNN & machine learning algorithms

We conduct gene mutation rate estimations via developing mutual information and Ewens sampling based convolutional neural network (CNN) and machine learning algorithms. More precisely, we develop a systematic methodology through constructing a CNN. Meanwhile, we develop two machine learning algorithms to study protein production with target gene sequences and protein structures. The core of the CNN and machine learning approach is to address a two-stage optimization problem to balance gene mutation rates during protein production. To wit, we try to optimally coordinate the consistency between the given input DNA sequences and the given (or optimally computed) target ones through controlling their intermediate gene mutation rates. The purposes in doing so are aimed to conduct gene editing and protein structure prediction. For example, after the gene mutation rates are estimated, the computing complexity of protein structure prediction will be reduced to a reasonable degree. Our developed CNN numerical optimization scheme consists of two newly designed machine learning algorithms. The stochastic gradients for the two algorithms are designed according to the Kuhn-Tucker conditions with boundary constraints and with the support of Ewens sampling, multi-input multi-output (MIMO) mutual information, and codon optimization techniques. The associated learning rate bounds are explicitly derived from the method and the two algorithms are numerically implemented. The convergence and optimality of the algorithms are mathematically proved. To illustrate the usage of our study, we also conduct a real-world data implementation.

Gene Editing for Enhanced Swine Production: Current Advances and Prospects

Traditional pig breeding has improved production traits but faces limitations in genetic diversity, disease resistance, and environmental adaptation. Gene editing technologies, such as CRISPR/Cas9, base editing, and prime editing, enable precise genetic modifications, overcoming these limitations and expanding applications to biomedical research. Here, we reviewed the advancements in gene editing technologies in pigs and explored pathways toward optimized swine genetics for a resilient and adaptive livestock industry. This review synthesizes recent research on gene editing tools applied to pigs, focusing on CRISPR/Cas9 and its derivatives. It examines their impact on critical swine production traits and their role as human disease models. Significant advancements have been made in targeting genes for disease resistance, such as those conferring immunity to porcine reproductive and respiratory syndrome viruses. Additionally, gene-edited pigs are increasingly used as models for human diseases, demonstrating the technology’s broader applications. However, challenges such as off-target effects, ethical concerns, and varying regulatory frameworks remain. Gene editing holds substantial potential for sustainable and productive livestock production by enhancing key traits and supporting biomedical applications. Addressing technical and ethical challenges through integrated approaches will be essential to realize its full potential, ensuring a resilient, ethical, and productive livestock sector for future generations

Gene Editing and Effect of ANP32 Proteins Splicing Variant for Viral Replication: A Review

Gene Editing and Effect of ANP32 Proteins Splicing Variant for Viral Replication: A Review

Technological approaches for commercial production of functional food through fish farming: Opportunities and challenges

Functional foods and nutraceuticals have gained attention as a result of the emergence of several human health disorders, including abdominal, obesity, diabetes, cardiovascular diseases, and neurodegeneration. The components of functional foods influence the physiological system, which enhances human health and fights against illness. The global consumption of fish and fish products has increased in recent years, driven by the presence of omega-3 fatty acids, oligosaccharides, glucosamine, vitamins A, D, and E, as well as minerals such as magnesium, potassium, copper, sulfur, zinc, iodine, and proteins. It is widely acknowledged that functional food ingredients are important for promoting health, lowering the risk of disease, and lowering medical expenses. Fish farming has emerged as a source of novel functional ingredients from marine resources for functional food as well as for therapeutics. The review paper will also cover various technological approaches such as CRISPR-Cas9 mediated gene editing, zinc finger nucleases, and TALENs for enhanced production of bioactive compounds from genetically modified fishes for commercial production of functional food as well as nutraceuticals. Keywords: Fish farming, Zinc finger nuclease, CRISPR/Cas9, Functional food, Nutraceuticals, Omega-3 fatty acids

De Novo Synthesis of Reticuline and Taxifolin Using Re-engineered Homologous Recombination in Yarrowia lipolytica.

Yarrowia lipolytica has been widely engineered as a eukaryotic cell factory to produce various important compounds. However, the difficulty of gene editing and the lack of efficient neutral sites make rewiring of Y. lipolytica metabolism challenging. Herein, a Cas9 system was established to redesign the Y. lipolytica homologous recombination system, which caused a more than 56-fold increase in the HR efficiency. The fusion expression of the hBrex27 sequence in the C-terminus of Cas9 recruited more Rad51 protein, and the engineered Cas9 decreased NHEJ, achieving 85% single-gene positive efficiency and 25% multigene editing efficiency. With this system, neutral sites on different chromosomes were characterized, and a deep learning model was developed for gRNA activity prediction, thus providing the corresponding integration efficiency and expression intensity. Subsequently, the tool and platform strains were validated by applying them for the de novo synthesis of (S)-reticuline and (2S)-taxifolin. The developed platform strains and tools helped transform Y. lipolytica into an easy-to-operate model cell factory, similar to Saccharomyces cerevisiae.

Advancing vegetable genetics with gene editing: a pathway to food security and nutritional resilience in climate-shifted environments.

As global populations grow and climate change increasingly disrupts agricultural systems, ensuring food security and nutritional resilience has become a critical challenge. In addition to grains and legumes, vegetables are very important for both human and animals because they contain vitamins, minerals, and fibre. Enhancing the ability of vegetables to withstand climate change threats is essential; however, traditional breeding methods face challenges due to the complexity of the genomic clonal multiplication process. In the postgenomic era, gene editing (GE) has emerged as a powerful tool for improving vegetables. GE can help to increase traits such as abiotic stresstolerance, herbicide tolerance, and disease resistance; improve agricultural productivity; and improve nutritional content and shelf-life by fine-tuning key genes. GE technologies such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR-Cas9) have revolutionized vegetable breeding by enabling specific gene modifications in the genome. This review highlights recent advances in CRISPR-mediated editing across various vegetable species, highlighting successful modifications that increase their resilience to climatic stressors. Additionally, it explores the potential of GE to address malnutrition by increasing the nutrient content of vegetable crops, thereby contributing to public health and food system sustainability. Additionally, it addresses the implementation of GE-guided breeding strategies in agriculture, considering regulatory, ethical, and public acceptance issues. Enhancing vegetable genetics via GE may provide a reliable and nutritious food supply for an expanding global population under more unpredictable environmental circumstances.

Peptide nucleic acids: Recent advancements and future opportunities in biomedical applications.

Peptide nucleic acids (PNA), synthetic molecules comprising a peptide-like backbone and natural and unnatural nucleobases, have garnered significant attention for their potential applications in gene editing and other biomedical fields. The unique properties of PNA, particularly enhanced stability/specificity/affinity towards targeted DNA and RNA sequences, achieved significant attention recently for gene silencing, gene correction, antisense therapy, drug delivery, biosensing and other various diagnostic aspects. This review explores the structure, properties, and potential of PNA in transforming genetic engineering including potent biomedical challenges. In Addition, we explore future perspectives and potential limitations of PNA-based technologies, highlighting the need for further research and development to fully realize their therapeutic and biotechnological potential.

SYNCAS based CRISPR-Cas9 editing in predatory mites, whiteflies and stinkbugs

Despite the establishment of CRISPR-Cas9 gene editing protocols in a wide range of organisms, genetic engineering is still challenging for many organisms due to constraints including lethality of embryo injection, difficulties in egg/embryo collection or viviparous lifestyles. Recently, an efficient CRISPR-Cas9 method, termed SYNCAS, was developed to genetically modify spider mites and thrips species. The method is based on maternal injection of formulated CRISPR-Cas9 using saponin and BAPC. Here, we investigate whether the method can be used to perform gene editing in other arthropods such as the beneficial predatory mites Amblyseius swirskii and Phytoseiulus persimilis, and the pests Bemisia tabaci and Nezara viridula. For the predatory mites, Antp and SLC25A38 were used as target genes, while the ortholog of the Drosophila melanogaster ABCG transporter white was targeted in B. tabaci and N. viridula. All species were successfully edited with the highest efficiencies (up to 39%) being obtained for B. tabaci. For A. swirskii and P. persimilis no clear phenotypes could be observed, even though SLC25A38 was successfully knocked-out. The lack of a color phenotype in SLC25A38 mutants was confirmed in the spider mite Tetranychus urticae. Disruption of the target gene Antp is likely lethal in predatory mites, as no true null mutants could be recovered. For B. tabaci, KO of white resulted in orange eyes which diverges from the phenotype seen in white mutants of D. melanogaster. In the last species, N. viridula, a single phenotypic mutant could be detected having a patchy white body coloration with wild type eye coloration. Genotyping revealed a single amino acid deletion at the target site, suggesting the creation of a hypomorphic allele. To conclude, the protocols provided in this work can contribute to the genetic study of predatory mites used in biological control, as well as hemipteran pests.

Translational Approach using Advanced Therapy Medicinal Products for Huntington's Disease.

Current therapeutic approaches for Huntington's disease (HD) focus on symptomatic treatment. Therefore, the unavailability of efficient disease-modifying medicines is a significant challenge. Regarding the molecular etiology, targeting the mutant gene or advanced translational steps could be considered promising strategies. The evidence in gene therapy suggests various molecular techniques, including knocking down mHTT expression using antisense oligonucleotides and small interfering RNAs and gene editing with zinc finger proteins and CRISPR-Cas9-based techniques. Several post-transcriptional and post-translational modifications have also been proposed. However, the efficacy and long-term side effects of these modalities have yet to be verified. Currently, cell therapy can be employed in combination with conventional treatment and could be used for HD in which the structural and functional restoration of degenerated neurons can occur. Several animal models have been established recently to develop cell-based therapies using renewable cell sources such as embryonic stem cells, induced pluripotent stem cells, mesenchymal stromal cells, and neural stem cells. These models face numerous challenges in translation into clinics. Nevertheless, investigations in Advanced Therapy Medicinal Products (ATMPs) open a promising window for HD research and their clinical application. In this study, the ATMPs entry pathway in HD management was highlighted, and their advantages and disadvantages were discussed.

Efficient promoter editing of the SBEIIb gene enables fine-tuning of the resistant starch content in rice.

Rice, a staple in diets, undergoes digestion post-consumption, often triggering a swift surge in blood sugar among diabetics, intensifying their health burden. Notably, resistant starch (RS) emerges as a potent ally in fostering satiety and mitigating metabolic syndrome in diabetes. The SBEIIb gene, a key orchestrator of starch branching enzymes, plays a pivotal role in starch synthesis, and its genetic alteration can dramatically boost RS content in rice. Cultivating RS-rich functional germplasms is of great significance for improving human nutrition and health. In this study, the application of CRISPR/Cas9 technology was investigated for precise multi-target editing within the SBEIIb promoter, aiming to generate valuable germplasm resources enriched with RS. The results revealed extensive mutations in the SBEIIb promoter region, resulting in a varied reduction in SBEIIb expression levels. Remarkably, the grains from the homozygous T1 mutant lines displayed a substantial elevation in RS content, ranging from 3 to 12 times greater than that of the wild-type, accompanied by notable alterations in the starch granule morphology of these grains. This study presents an effective breeding strategy for the precise enhancement of RS content and establishes a foundation for further insights into the molecular mechanisms governing cis-element regulation of RS synthesis.

Gene editing of CD3 epsilon gene to redirect regulatory T cells for adoptive T cell transfer

Gene editing of CD3 epsilon gene to redirect regulatory T cells for adoptive T cell transfer

Focused ultrasound widely broadens AAV-delivered Cas9 distribution and activity.

Because children have little temporal exposure to environment and aging, most pediatric neurological diseases are inherent, i.e. genetic. Since postnatal neurons and astrocytes are mostly non-replicating, gene therapy and genome editing present enormous promise in child neurology. Unlike in other organs, which are highly permissive to adeno-associated viruses (AAV), the mature blood-brain barrier (BBB) greatly limits circulating AAV distribution to the brain. Intrathecal administration improves distribution but to no more than 20% of brain cells. Focused ultrasound (FUS) opens the BBB transiently and safely. In the present work we opened the hippocampal BBB and delivered a Cas9 gene via AAV9 intrathecally. This allowed brain first-pass, and subsequent vascular circulation and re-entry through the opened BBB. The mouse model used was of Lafora disease, a neuroinflammatory disease due to accumulations of misshapen overlong-branched glycogen. Cas9 was targeted to the gene of the glycogen branch-elongating enzyme glycogen synthase. We show that FUS dramatically (2000-fold) improved hippocampal Cas9 distribution and greatly reduced the pathogenic glycogen accumulations and hippocampal inflammation. FUS is in regular clinical use for other indications. Our work shows that it has the potential to vastly broaden gene delivery or editing along with clearance of corresponding pathologic basis of brain disease.

Improving blood-brain barrier penetration in Hurler syndrome using an IDUA-ApoE fusion enzyme delivered via the PS Gene Editing System

Improving blood-brain barrier penetration in Hurler syndrome using an IDUA-ApoE fusion enzyme delivered via the PS Gene Editing System

Galectin-3 secreted by triple-negative breast cancer cells regulates T cell function.

Triple-negative breast cancer (TNBC) is an aggressive subtype that accounts for 10-15 % of breast cancer. Current treatment of high-risk early-stage TNBC includes neoadjuvant chemo-immune therapy. However, the substantial variation in immune response prompts an urgent need for new immune-targeting agents. This requires a comprehensive understanding of TNBC's tumor microenvironment. We recently demonstrated that Galectin-3 (Gal-3) binding protein/Gal-3 complex secreted by TNBC cells induces immunosuppression, through inhibiting CD45 signaling in T cells. Here, we further investigated the interaction between secreted Gal-3 and T cells in TNBC. Using CRISPR/Cas9 gene editing of the TNBC MDA-MB-231 cell-line, we obtained Gal-3 negative(neg) clones. We studied these in an in-vitro model, co-cultured with peripheral blood mononuclear cells (PBMC) to imitate immune-tumor interaction, and in an in-vivo model, when implanted in mice. Gal-3neg tumors in mice had decelerated tumor growth after PBMC inoculation. In contrast, the Gal-3 positive(pos) tumors continued growing despite PBMC inoculation, and tumor T regulatory cell (CD4/FoxP3+) infiltration increased. RNA sequencing of T cells from women with TNBC with elevated plasma levels of Gal-3 revealed significantly lower expression of oxidative phosphorylation genes than in T cells from healthy women. Similarly, in our in-vitro model, the decreased expression of oxidative phosphorylation genes and mitochondrial dysfunction resulted in a significant increase in CD8 intracellular reactive oxygen species. Consequently, T exhausted cells (CD8/PD1/Tim3/Lag3+) significantly increased in PBMC co-cultured with Gal-3pos TNBCs. To conclude, we revealed a novel TNBC-related Gal-3 suppressor mechanism that involved upregulation of CD4 T regulatory and of CD8 T exhausted cells.

A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9.

CRISPR-based base editor (BE) offer diverse editing options for genetic engineering of microorganisms, but its application is limited by protospacer adjacent motif (PAM) sequences, context preference, editing window, and off-target effects. Here, a series of iteratively improved cytosine base editors (CBEs) are constructed using the FrCas9 nickase (FrCas9n) with the unique PAM palindromic structure (NNTA) to alleviate these challenges. The deaminase domain-inlaid FrCas9n exhibits an editing range covering 38 nucleotides upstream and downstream of the palindromic PAM, without context preference, which is 6.3 times larger than that of traditional CBEs. Additionally, lower off-target editing is achieved when incorporating high-fidelity mutations at R61A and Q964A in FrCas9n, while maintaining high editing efficiency. The final CBE, HF-ID824-evoCDA-FrCas9n demonstrates broad applicability across different microbes such as Escherichia coli MG1655, Shewanella oneidensis MR-1, and Pseudomonas aeruginosa PAO1. Collectively, this tool offers robust gene editing for facilitating mechanistic studies, functional exploration, and protein evolution in microbes.