Gene Editing Research Articles

The application value assessment of CRISPR in screening and optimizing bacteria for industrial production

CRISPR is a gene editing technology derived from bacteria and has been widely used in the current biological field. However, the use range of it is still mainly in laboratories. The industrial production field can rarely see its role, such as the screening and optimization of production strains. This article compares the advantages and disadvantages of CRISPR with traditional natural sampling or mutagenesis breeding combined with selective culture medium screening technology in actual production in technical and non-technical fields, emphasizing that CRISPR technology has great advantages in integrating excellent traits and improving breeding efficiency. However, it is still slightly inferior to traditional technology in terms of breeding costs, experimental conditions, and social acceptance. The significance of this study lies in a relatively comprehensive analysis of the various indicators worth comparing when CRISPR technology is actually applied to industrial breeding, which can provide a certain reference value for whether the technology should be officially put into industrial use. However, there are still many defects of CRISPR technology which is difficult to correct; although the direction of its improvement is fairly clear, its never easy to work, and the development will still need some time.

Targeting EBV Episome for Anti-Cancer Therapy: Emerging Strategies and Challenges.

As a ubiquitous human pathogen, the Epstein-Barr virus (EBV) has established lifelong persistent infection in about 95% of the adult population. The EBV infection is associated with approximately 200,000 human cancer cases and 140,000 deaths per year. The presence of EBV in tumor cells provides a unique advantage in targeting the viral genome (also known as episome), to develop anti-cancer therapeutics. In this review, we summarize current strategies targeting the viral episome in cancer cells. We also highlight emerging technologies, such as clustered regularly interspersed short palindromic repeat (CRISPR)-based gene editing or activation, which offer promising avenues for selective targeting of the EBV episome for anti-cancer therapy. We discuss the challenges, limitations, and future perspectives associated with these strategies, including potential off-target effects, anti-cancer efficacy and safety.

CRISPR/Cas9-Enhanced CAR-T Cell Therapy for Hematological Malignancies

CRISPR/Cas9 technology has brought revolution to the field of gene editing, offering precise and efficient tools for genetic modifications. One of its most promising applications lies in improving CAR-T cell therapy for treating hematological malignancies. Among approaches to treating blood cancers, CAR-T therapy, which programs T cells to recognize and eliminate cancer cells, has shown some success for B-cell malignancies such as diffuse large B-cell lymphoma (DLBCL) and B-cell acute lymphoblastic leukemia (ALL). However, challenges such as immune evasion, cytokine release syndrome (CRS), neurotoxicity, and limited CAR-T persistence remain significant barriers. CRISPR/Cas9 can optimize CAR-T therapy by precisely inserting CAR genes into specific loci, knocking out inhibitory genes like PD-1 to enhance persistence, and enabling multi-targeting strategies to overcome tumor immune escape. Clinical trials demonstrate the feasibility and potential of CRISPR-edited CAR-T cells, showing improved safety, durability, and efficacy. This study explores the synergistic application of CRISPR/Cas9 in CAR-T therapy, addressing its current limitations and providing a pathway to safer and more effective treatments for hematological malignancies

A matter of right or wrong: Divisive attributes of moralized science and technology attitudes.

In recent years, scholars have theorized that one factor enflaming public divides over science and technology is moralization: an individual's perception that their position on an issue is rooted in fundamental moral right and wrong. In this article, I provide evidence for this proposition across five pre-registered hypotheses about the divisive attributes of moralized attitudes in the context of science and technology. Using public opinion data in the United States on three issues-combating climate change, developing gene editing therapies for humans, and labeling genetically modified food-this study demonstrates that moralized attitudes have the potential to exacerbate resistance to scientific evidence and hostility between those with opposing positions. These findings provide strong proof of concept that studying variation in the degree to which individuals moralize issues is an important future direction for understanding persistent public divides over science and technology.

Harnessing the Streptomyces-originating type I-E CRISPR/Cas system for efficient genome editing in Streptomyces.

Since their discovery, CRISPR/Cas systems have significantly expanded the genetic toolbox, aiding in the exploration and enhanced production of natural products across various microbes. Among these, class 2 CRISPR/Cas systems are simpler and more broadly used, but they frequently fail to function effectively in many Streptomyces strains. In this study, we present an engineered class 1 type I CRISPR/Cas system derived from Streptomyces avermitilis, which enables efficient gene editing in phylogenetically distant Streptomyces strains. Through a plasmid interference assay, we identified the effective protospacer adjacent motif as 5'-AAN-3'. Utilizing this system, we achieved targeted chromosomal deletions ranging from 8 bp to 100 kb, with efficiencies exceeding 92%. We further utilized this system to insert DNA fragments into different Streptomyces genomes, facilitating the heterologous expression of exogenous genes and the activation of endogenous natural product biosynthetic gene clusters. Overall, we established a type I CRISPR/Cas-based gene-editing methodology that significantly advances the exploration of Streptomyces, known for their rich natural product resources. This is the first report of a gene editing tool developed based on the endogenous class 1 type I CRISPR/Cas system in Streptomyces spp. Our work enriches the Streptomyces gene manipulation toolbox and advances the discovery of valuable natural products within these organisms.

A novel susceptibility locus to Erysiphe necator (SEN2) identified by genetic mapping of automated microscopy computer vision data in grapevines.

Powdery mildew, caused by the fungus Erysiphe necator, is one of the primary causes of grape yield loss across the globe. While numerous resistance loci have been identified in various grapevine species, the genetic determinants of susceptibility to E. necator remain largely unexplored. Understanding the genetics of susceptibility for pathogenesis is equally important for developing durable resistance grapevines against this pathogen. To identify these factors in Vitis interspecific hybrid 'Chambourcin', a controlled leaf disc assay was conducted for two years using an automated microscopy phenotyping system with 273 F1 genotypes from a cross of 'Chambourcin' and V. vinifera 'Cabernet Sauvignon'. Additionally, a high-resolution linkage map using the same number of 'Chambourcin'-derived hybrids was constructed with 355 simple sequence repeats (SSR) and 1,394 RNaseH2-dependent amplicon sequencing (rhAmpSeq)-derived haplotype markers that clustered into 19 linkage groups. A quantitative trait locus (QTL) analysis identified a susceptibility locus (here named Sen2) on linkage group (LG) 7 explaining 8.90 % - 12.57 % of the total phenotypic variance. The markers associated with this susceptibility locus were used to identify 78 accessions in the USDA-ARS cold hardy Vitis collection at Geneva, NY that carry Sen2 and can be used to selectively exclude susceptible progenies. Additionally, 6 accessions carry the alternative haplotype encoding recessive resistance and can be used for resistance breeding. The identification of powdery mildew susceptibility loci is crucial for identifying genes that could be targeted for gene knock-out, gene editing, RNA interference (RNAi), or selection among breeding genotypes to enhance sustainable protection against pathogens.

Mutagenesis and analysis of contrasting wheat lines do not support a role for PFT in Fusarium head blight resistance.

Ambiguity about whether the histidine-rich calcium-binding protein-coding gene (HisR) or the pore-forming toxin-like gene (PFT) or both are responsible for Fusarium head blight (FHB) resistance conferred by the Fhb1 quantitative trait locus hinders progress toward elucidating Fhb1 resistance mechanisms. Here, with a series of developed lines with or without PFT but all possessing HisR and five HisR-carrying PFT mutant lines created via gene editing, we show that PFT does not confer FHB resistance and that the HisR resistance effect does not require PFT in the tested conditions. We also show that PFT mutations are not associated with morphological and phenological characteristics that often affect FHB severity.

Revisiting the female germline cell development.

The formation of the female germline is the fundamental process in most flowering plants' sexual reproduction. In Arabidopsis, only one somatic cell obtains the female germline fate, and this process is regulated by different pathways. Megaspore mother cell (MMC) is the first female germline, and understanding MMC development is essential for comprehending the complex mechanisms of plant reproduction processes. Recently, more advanced technologies such as whole-mount single-molecule fluorescence in situ hybridization (smFISH), laser-assisted microdissection (LCM), chromatin immunoprecipitation/sequencing, and CRISPR gene editing have provided opportunities to reveal the mechanism of female germline development at different stages. Single-cell transcriptome/spatial transcriptomics analysis helps to investigate complex cellular systems at the single-cell level, reflecting the biological complexity of different cell types. In this review, we highlight recent progress that facilitates the development of the female germline to explore the roles of crucial gene regulatory networks, epigenetic pathways, cell-cycle regulators, and phytohormones in this process. This review discusses three key phases in female germline development and provides the possibility of distinct pathways restricting germline development in the future.

Activation of three targets by a TAL effector confers susceptibility to bacterial blight of cotton

Bacterial transcription activator-like effectors (TALEs) promote pathogenicity by activating host susceptibility (S) genes. To understand the pathogenicity and host adaptation of Xanthomonas citri pv. malvacearum (Xcm), we assemble the genome and the TALE repertoire of three recent Xcm Texas isolates. A newly evolved TALE, Tal7b, activates GhSWEET14a and GhSWEET14b, different from GhSWEET10 targeted by a TALE in an early Xcm isolate. Activation of GhSWEET14a and GhSWEET14b results in water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identify a pectin lyase gene as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9 gene editing supports the function of GhSWEETs in cotton bacterial blight and the promise of disrupting the TALE-binding site in S genes for disease management. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes to promote pathogenicity.

CRISPR-Cas9 Targeting PCSK9: A Promising Therapeutic Approach for Atherosclerosis.

CRISPR-Cas9 gene editing technology, as an innovative biomedical tool, holds significant potential in the prevention and treatment of atherosclerosis. By precisely editing key genes such as PCSK9, CRISPR-Cas9 offers the possibility of long-term regulation of low-density lipoprotein cholesterol (LDL-C), which may reduce the risk of cardiovascular diseases. Early clinical studies of gene editing therapies like VERVE-101 have yielded encouraging results, highlighting both the feasibility and potential efficacy of this technology. However, clinical applications still face challenges such as off-target effects, immunogenicity, and long-term safety. Future research should focus on enhancing the specificity and efficiency of gene editing, optimizing delivery systems, and improving personalized treatment strategies. Additionally, the establishment of ethical and legal regulatory frameworks will be critical for the safe adoption of this technology. With the continued advancement of gene editing technology, CRISPR-Cas9 may become an important tool for treating atherosclerosis and other complex diseases.

The Evolution of Immunosuppressive Therapy in Pig-to-Nonhuman Primate Organ Transplantation.

An overview is provided of the evolution of strategies towards xenotransplantation during the past almost 40 years, focusing on advances in gene-editing of the organ-source pigs, pre-transplant treatment of the recipient, immunosuppressive protocols, and adjunctive therapy. Despite initial challenges, including hyperacute rejection resulting from natural (preformed) antibody binding and complement activation, significant progress has been made through gene editing of the organ-source pigs and refinement of immunosuppressive regimens. Major steps were the identification and deletion of expression of the three known glycan xenoantigens on pig vascular endothelial cells, the transgenic expression of human "protective" proteins, e.g., complement-regulatory, coagulation-regulatory, and anti-inflammatory proteins, and the administration of an immunosuppressive regimen based on blockade of the CD40/CD154T cell co-stimulation pathway. Efforts to address systemic inflammation followed. The synergy between gene editing and judicious immunomodulation appears to largely prevent graft rejection and is associated with a relatively good safety profile. Though there remains an incidence of severe or persistent proteinuria (nephrotic syndrome) in a minority of cases. This progress offers renewed hope for patients in need of life-saving organ transplants.

Characterization of the role of spatial proximity of DNA double-strand breaks in the formation of CRISPR-Cas9-induced large structural variations

Structural variations (SVs) play important roles in genetic diversity, evolution, and carcinogenesis and are, as such, important for human health. However, it remains unclear how spatial proximity of double-strand breaks (DSBs) affects the formation of SVs. To investigate if spatial proximity between two DSBs affects DNA repair, we used data from 3C experiments (Hi-C, ChIA-PET, and ChIP-seq) to identify highly interacting loci on six different chromosomes. The target regions correlate with the borders of mega-base sized Topologically Associated Domains (TADs), and we used CRISPR-Cas9 nuclease and pairs of single guide RNAs (sgRNAs) against these targets to generate DSBs in both K562 cells and H9 human embryonic stem cells (hESC). Droplet Digital PCR (ddPCR) was used to quantify the resulting recombination events, and high-throughput sequencing was used to analyze the chimeric junctions created between the two DSBs. We observe a significantly higher formation frequency of deletions and inversions with DSBs in proximity as compared to deletions and inversions with DSBs not in proximity in K562 cells. Additionally, our results suggest that DSB proximity may affect the ligation of chimeric deletion junctions. Taken together, spatial proximity between DSBs is a significant predictor of large-scale deletion and inversion frequency induced by CRISPR-Cas9 in K562 cells. This finding has implications for understanding SVs in the human genome and for the future application of CRISPR-Cas9 in gene editing and the modelling of rare SVs.

Differential roles of human CD4+ and CD8+ regulatory T cells in controlling self-reactive immune responses.

Here we analyzed the relative contributions of CD4+ regulatory T cells expressing Forkhead box protein P3 (FOXP3) and CD8+ regulatory T cells expressing killer cell immunoglobulin-like receptors to the control of autoreactive T and B lymphocytes in human tonsil-derived immune organoids. FOXP3 and GZMB respectively encode proteins FOXP3 and granzyme B, which are critical to the suppressive functions of CD4+ and CD8+ regulatory T cells. Using CRISPR-Cas9 gene editing, we were able to achieve a reduction of ~90-95% in the expression of these genes. FOXP3 knockout in tonsil T cells led to production of antibodies against a variety of autoantigens and increased the affinity of influenza-specific antibodies. By contrast, GZMB knockout resulted in an increase in follicular helper T cells, consistent with the ablation of CD8+ regulatory T cells observed in mouse models, and a marked expansion of autoreactive CD8+ and CD4+ T cells. These findings highlight the distinct yet complementary roles of CD8+ and CD4+ regulatory T cells in regulating cellular and humoral responses to prevent autoimmunity.

RNA-Targeting CRISPR/CasRx system relieves disease symptoms in Huntington’s disease models

BackgroundHD is a devastating neurodegenerative disorder caused by the expansion of CAG repeats in the HTT. Silencing the expression of mutated proteins is a therapeutic direction to rescue HD patients, and recent advances in gene editing technology such as CRISPR/CasRx have opened up new avenues for therapeutic intervention.MethodsThe CRISPR/CasRx system was employed to target human HTT exon 1, resulting in an efficient knockdown of HTT mRNA. This therapeutic effect was substantiated in various models: HEK 293 T cell, the HD 140Q-KI mouse, and the HD-KI pig model. The efficiency of the knockdown was analyzed through Western blot and RT-qPCR. Additionally, neuropathological changes were examined using Western blot, immunostaining, and RNA sequencing. The impact on motor abilities was assessed via behavioral experiments, providing a comprehensive evaluation of the treatment's effectiveness.ResultsCRISPR/CasRx system can significantly reduce HTT mRNA levels across various models, including HEK 293 T cells, HD 140Q-KI mice at various disease stages, and HD-KI pigs, and resulted in decreased expression of mHTT. Utilizing the CRISPR/CasRx system to knock down HTT RNA has shown to ameliorate gliosis in HD 140Q-KI mice and delay neurodegeneration in HD pigs.ConclusionsThese findings highlight the effectiveness of the RNA-targeting CRISPR/CasRx as a potential therapeutic strategy for HD. Furthermore, the success of this approach provides valuable insights and novel avenues for the treatment of other genetic disorders caused by gene mutations.

Enhancing precision in cancer treatment: the role of gene therapy and immune modulation in oncology.

Gene therapy has long been a cornerstone in the treatment of rare diseases and genetic disorders, offering targeted solutions to conditions once considered untreatable. As the field advances, its transformative potential is now expanding into oncology, where personalized therapies address the genetic and immune-related complexities of cancer. This review highlights innovative therapeutic strategies, including gene replacement, gene silencing, oncolytic virotherapy, CAR-T cell therapy, and CRISPR-Cas9 gene editing, with a focus on their application in both hematologic malignancies and solid tumors. CRISPR-Cas9, a revolutionary tool in precision medicine, enables precise editing of cancer-driving mutations, enhancing immune responses and disrupting tumor growth mechanisms. Additionally, emerging approaches target ferroptosis-a regulated, iron-dependent form of cell death-offering new possibilities for selectively inducing tumor cell death in resistant cancers. Despite significant breakthroughs, challenges such as tumor heterogeneity, immune evasion, and the immunosuppressive tumor microenvironment (TME) remain. To overcome these barriers, novel approaches like dual-targeting, armored CAR-T cells, and combination therapies with immune checkpoint inhibitors and ferroptosis inducers are being explored. Additionally, the rise of allogeneic "off-the-shelf" CAR-T therapies offers scalable and more accessible treatment options. The regulatory landscape is evolving to accommodate these advancements, with frameworks like RMAT (Regenerative Medicine Advanced Therapy) in the U.S. and ATMP (Advanced Therapy Medicinal Products) in Europe fast-tracking the approval of gene therapies. However, ethical considerations surrounding CRISPR-based gene editing-such as off-target effects, germline editing, and ensuring equitable access-remain at the forefront, requiring ongoing ethical oversight. Advances in non-viral delivery systems, such as lipid nanoparticles (LNPs) and exosomes, are improving the safety and efficacy of gene therapies. By integrating these innovations with combination therapies and addressing regulatory and ethical concerns, gene therapy is poised to revolutionize cancer treatment, providing durable, effective, and personalized solutions for both hematologic and solid tumors.

Loss of Myostatin Affects m6A Modification but Not Semen Characteristics in Bull Spermatozoa.

N6-methyladenosine (m6A) modification is a key methylation modification involved in reproductive processes. Myostatin gene editing (MT) in cattle is known to enhance muscle mass and productivity. However, the changes in m6A modification in MT bull sperm remain poorly understood. In the MT and wild-type (WT) groups, we identified 25,542 and 22,253 m6A peaks, respectively, mainly concentrated in the coding sequence (CDS) and 3' untranslated region (UTR) of genes. The MT group showed an increase in gene transcription, but there was no significant difference in the overall m6A peaks pattern. There was also no significant difference in m6A motif and chromosome distribution between MT and WT groups. Most genes had less m6A modification sites. A total of 1120 m6A peaks were significantly different, corresponding to 1053 differentially m6A-methylated genes (DMMGs). These DMMGs are mainly associated with G protein-coupled receptor signaling pathways and the overall composition of the cell membrane. Furthermore, an MCL clustering analysis of 111 differentially m6A-methylated and expressed genes identified seven key genes (RHOA, DAAM1, EXOC4, GNA12, PRICKLE1, SCN1A, and STXBP5L), with the cytoskeleton and migration-related gene, RHOA, being the most important gene located at the center of the gene network. However, the analysis of sperm morphology and motility indicated no significant changes in semen volume, sperm count, sperm viability, plasma membrane integrity, acrosome membrane integrity, or mitochondrial membrane integrity. This study provides a map of m6A methylation in spermatozoa from MT and WT bulls, identifies key differential m6A genes that are affected by the myostatin gene but do not affect sperm morphology and viability in MT bulls, and provides a theoretical basis for the breeding quality of MT bulls.

Cascade-Responsive Nanoparticles for Efficient CRISPR/Cas9-Based Glioblastoma Gene Therapy.

CRISPR/Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing technology represents great promise for treating glioblastoma (GBM) due to its potential to permanently eliminate tumor pathogenic genes. Unfortunately, delivering CRISPR to the GBM in a safe and effective manner is challenging. Herein, a glycosylated and cascade-responsive nanoparticle (GCNP) that can effectively cross the blood-brain barrier (BBB) and activate CRISPR/Cas9-based gene editing only in the GBM is designed. The GCNP possesses a cationic polyplex core and a glycosylated polymer layer that is capable of cascading response to low pH and high GSH concentration, so that the release of CRISPR/Cas9 only takes place after crossing the BBB and entering the GBM where the acidic tumor microenvironment and high concentration of glutathione (GSH) are present. By targeting the programmed death-ligand 1 (PD-L1) in GBM, GCNP effectively inhibited the tumor growth and greatly prolonged the survival time of GBM-bearing mice when combined with temozolomide (TMZ).

Tissue-specific modulation of CRISPR activity by miRNA-sensing guide RNAs.

Nucleic acid nanostructures offer unique opportunities for biomedical applications due to their sequence-programmable structures and functions, which enable the design of complex responses to molecular cues. Control of the biological activity of therapeutic cargoes based on endogenous molecular signatures holds the potential to overcome major hurdles in translational research: cell specificity and off-target effects. Endogenous microRNAs (miRNAs) can be used to profile cell type and cell state, and are ideal inputs for RNA nanodevices. Here, we present CRISPR MiRAGE (miRNA-activated genome editing), a tool comprising a dynamic single-guide RNA that senses miRNA complexed with Argonaute proteins and controls downstream CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) activity based on the detected miRNA signature. We study the operation of the miRNA-sensing single-guide RNA and attain muscle-specific activation of gene editing through CRISPR MiRAGE in models of Duchenne muscular dystrophy. By enabling RNA-controlled gene editing activity, this technology creates opportunities to advance tissue-specific CRISPR treatments for human diseases.

CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers

Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimize a bacterial CRISPR-hybrid system coupled with FACS, and identified high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allows effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.

Integration of Genetic Resistance Mechanisms in Sustainable Crop Breeding Programs-A Review

The integration of genetic resistance mechanisms into sustainable crop breeding programs is critical for addressing global agricultural challenges, including the increasing threats posed by pests, diseases, and climate change. Genetic resistance, which involves the use of innate plant defense mechanisms, provides an environmentally friendly alternative to chemical controls and plays a pivotal role in enhancing crop resilience. Advances in molecular biology, such as CRISPR-Cas9 gene editing and multi-omics technologies (genomics, transcriptomics, and metabolomics), have revolutionized resistance breeding, enabling the precise identification, modification, and deployment of resistance traits. These tools facilitate the development of crops with enhanced resistance to biotic and abiotic stresses while reducing yield penalties and linkage drag. Challenges such as the evolution of pathogen virulence, the breakdown of race-specific resistance genes, and the trade-offs between resistance and crop quality remain significant hurdles. Durable resistance, achieved by combining qualitative and quantitative resistance traits, offers a promising approach to mitigate these issues and delay resistance breakdown. Agroecological practices, such as crop diversification, companion planting, and organic amendments, can complement genetic resistance by reducing pathogen pressure and improving ecosystem stability. International research collaborations, such as those led by CGIAR, along with local capacity-building efforts, are essential to ensure the equitable dissemination of resistance technologies, particularly in resource-limited regions. Despite these advances, socioeconomic and regulatory barriers, including public skepticism toward genetically engineered crops and stringent approval processes for GMOs and gene-edited varieties, hinder widespread adoption. Increased investments in breeding research, streamlined regulatory frameworks, and policies promoting resistant varieties are vital to overcoming these challenges. As the global demand for food continues to rise amidst climate uncertainties, the integration of cutting-edge genetic tools, ecological principles, and collaborative efforts offers a pathway to more sustainable and resilient agricultural systems. By addressing current limitations and leveraging emerging technologies, genetic resistance can significantly contribute to global food security and the sustainability of modern farming practices.