Search Results (308)

Spinocerebellar ataxia (SCA), an autosomal dominant neurodegenerative condition, is marked by a gradual deterioration of cerebellar function. To date, more than 40 distinct SCA subtypes have been identified, with some attributed to CAG repeat expansions and others to point mutations or deletions. Among these, spinocerebellar ataxia type 14 (SCA14) stems from missense mutations or deletions within the PRKCG gene, encoding protein kinase C gamma (PKCγ), a pivotal signaling molecule abundant in Purkinje cells. Despite its significance, the precise mechanisms underlying how genetic alterations trigger Purkinje cell malfunction and degeneration remain elusive. Given the prominent role and high expression of PKCγ in Purkinje cells, SCA14 presents a unique opportunity to unravel the underlying pathogenesis. A straightforward hypothesis posits that alterations in the biological activity of PKCγ underlie the disease phenotype, and there are hints that mutated PKCγ proteins exhibit altered enzymatic function. Our prior research focused on the PKCγ-G118D mutation, commonly found in SCA14 patients, located in the regulatory domain of the protein. While cellular assays demonstrated enhanced enzymatic activity for PKCγ-G118D, transgenic mice carrying this mutation failed to exhibit suppressed dendritic development in cerebellar cultures, raising questions about its impact within living Purkinje cells. One hypothesis is that endogenous PKCγ might interfere with the expression or effect of PKCγ-G118D. To further investigate, we leveraged CRISPR-Cas9 technology to generate a PKCγ knockout mouse model and integrated it with an L7-based, Purkinje cell-specific transfection system to analyze the effects of G118D protein expression on the dendritic morphology of developing Purkinje cells. Our findings reveal that, utilizing this approach, PKCγ-G118D exerts a detrimental effect on Purkinje cell growth, confirming its negative influence, indicating that the potential of the G118D mutation to contribute to SCA14 pathogenesis. Full article

Cotton fiber length is an important measurement for application in the textile industry, and researchers are seeking to cultivate cotton plants with longer fibers. In this study, cotton fiber genes were systematically reviewed through meta-analysis in terms of extending and shortening fiber and the use of different research technologies for the first time. PubMed, Web of Science, China National Knowledge Infrastructure (CNKI), and Baidu Xueshu databases were included as literature retrieval sources. A total of 21,467 articles were retrieved, and 45 articles were used in the final analysis. Data analysis was performed using RevMan 5.4 software. To shorten cotton fiber length, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology was superior to virus-induced gene silencing (VIGS) technology and RNA interference (RNAi) technology [p = 0.002, MD = −1.05, 95% CI (−1.73, −0.37), Chi² = 39.89]. To increase cotton fiber length, CRISPR-Cas9 technology had a similar effect as VIGS technology [p = 0.12, MD = −0.59, 95% CI (−1.33, −0.15), Chi² = 0.17]. When some genes (GhLAC15, GhALDH7B4, GhMDHAR1A/GhDHAR2A, STTM-miR396b, GhMYB44, GhFP2, GhMYB7, GhKNL1, GhTCP4, GhHDA5, GhGalT1, GhKNOX6, GhXB38D, and GhBZR3) were damaged, cotton fiber length increased. Furthermore, we found that after gene interference, the fiber-shortening genes occurred more frequently than the fiber-elongating genes. Synergistic research on these genes may better promote cotton fiber elongation. Full article

High quality stands as a pivotal competitive edge in the rice industry. Optimizing amylose content (AC) and the physicochemical properties of endosperm starch by regulating the Wx gene is crucial for enhancing rice grain quality. In this study, we created a novel Wx^b-d25 allele by deleting a 25 bp segment (−26 to −2) within the Wx core promoter using CRISPR/Cas9. Compared with the wild type and the previously reported Wx^b-i1, Wx^b-d25 exhibited no significant changes in agronomic traits. However, its grains displayed temperature-dependent variations in AC and altered transparency and viscosity characteristics, holding the potential to synergistically improve both the eating and cooking quality (ECQ) and appearance quality (AQ) of rice. Further studies demonstrated that this promoter modification, by partially disrupting the transcription initiator, significantly downregulated the original Wx-01 transcript and generated a novel Wx transcript (ONT.7395.1) in Wx^b-d25 grains. Despite its low expression abundance, the ONT.7395.1 transcript could be completely processed into mature Wx mRNA. The combined effects of the dual transcripts resulted in significantly increased Wx gene expression and AC in Wx^b-d25 grains under conventional cultivation conditions. These findings provide a genetic resource and a theoretical foundation for utilizing the Wx^b-d25 allele to improve rice grain quality. Full article

CRISPR-associated endoribonucleases (Cas RNases) cleave single-stranded RNA in a highly sequence-specific manner by recognizing and binding to short RNA sequences known as direct repeats (DRs). Here, we investigate the potential of exploiting Cas RNases for the regulation of target genes with one or more DRs introduced into the 3′ untranslated region, an approach we refer to as DREDGE (direct repeat-enabled downregulation of gene expression). The DNase-dead version of Cas12a (dCas12a) was identified as the most efficient among five different Cas RNases tested and was subsequently evaluated in doxycycline-regulatable systems targeting either stably expressed fluorescent proteins or an endogenous gene. DREDGE performed superbly in stable cell lines, resulting in up to 90% downregulation with rapid onset, notably in a fully reversible and highly selective manner. Successful control of an endogenous gene with DREDGE was demonstrated in two formats, including one wherein both the DR and the transgene driving expression of dCas12a were introduced in one step by CRISPR-Cas. Our results establish DREDGE as an effective method for regulating gene expression in a targeted, highly selective, and fully reversible manner, with several advantages over existing technologies. Full article

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization, and in severe cases from vitamin B₆-responsive seizures. To study HPP, we generated alpl^-/- zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl^-/- compared to alpl^+/+ embryos. The survival of alpl^-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl^-/- embryos. B₆ vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl^-/- embryos. Accumulation of d3-pyridoxal 5′-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl^-/- embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B₆ metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl^-/- embryos. Metabolic profiling of alpl^-/- larvae using direct-infusion high-resolution mass spectrometry showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B₆-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated the first zebrafish model of HPP that shows multiple features of human disease and which is suitable for the study of the pathophysiology of HPP and for the testing of novel treatments. Full article

Background: Since 2011, re-emerging pseudorabies virus (PRV) variant strains have been widespread in swine herds immunized with the classical PRV vaccine in China, suggesting that it is necessary to develop a new vaccine against these PRV variant strains. Methods: Here, based on a PRV mutant strain isolated in Jinmen (JM), two recombinant strains were constructed using CRISPR/Cas9 technology, including PRV-JM-ΔEK with the deletion of the gE and TK genes and PRV-JM-ΔEI92K with the deletion of the gE, gI, US2, US9, and TK genes. Results: A one-step growth curve and plaque assay revealed that the cell-to-cell transmission ability of PRV-JM-ΔEI92K was lower than that of PRV-JM-ΔEK. However, the replication ability of PRV-JM-ΔEI92K was approximately 10 times higher than that of PRV-JM-ΔEK, similar to wild-type PRV-JM. The intramuscular injection of 10⁶ TCID₅₀ of PRV-JM-ΔEK or PRV-JM-ΔEI92K could not cause death in mice, and both could produce specific antibodies against gB and gD. The survival rate of mice immunized with both recombinant viruses was 100% when the mice were challenged by the PRV-JM strain. Histopathological sections from the PRV-JM-ΔEK group showed milder pathological changes compared to the PRV-JM-ΔEI92K group, proving that PRV-JM-ΔEK provided more effective protection. In pigs injected with 10⁶ TCID₅₀ of PRV-JM-ΔEK or PRV-JM-ΔEI92K, their body temperature did not rise, and their weight gain was not affected. Both recombinant viruses could induce the production of gB- and gD-specific antibodies and neutralizing antibodies. After the challenge of the PRV-JM virus, neutralizing antibody production was rapidly induced and lasted for at least 3 weeks. Pigs immunized with both PRV-JM-ΔEI92K and PRV-JM-ΔEK had a 100% survival rate, demonstrating that both recombinant viruses could provide effective protection. Conclusions: Compared with PRV-JM-ΔEK, PRV-JM-ΔEI92K had better safety. In conclusion, we constructed two PRV recombinant viruses, which have the potential to be used as a live carrier vaccine. Full article

Fel d1 is the most important allergen secreted by cats, which can trigger asthma in sensitive individuals. Our objective was to knock-out the Fel d1 gene in the fetal fibroblasts of cats through CRISPR–Cas9 technology with two sgRNAs and to determine the impact of such mutations on the antigenicity of the Fel d1 protein. DNA samples from 38 domestic cats were collected and amplified by PCR to obtain the complete sequence of the Fel d1 gene. Throughout evolution, Fel d1 polypeptide chain 1(CH1) has proven to be much more conserved than Fel d1 polypeptide chain 2(CH2); therefore, we targeted CH2 and designed two single-guide RNAs (CH2-sgRNA-1 and CH2-sgRNA-2) for this region. Using these constructed sgRNAs, we performed gene knock-out in fetal fibroblasts, resulting in two mutations within the target gene. Following this, DNA was extracted and the target site product was cloned using TA cloning via PCR, and a single colony from this process was sequenced to analyze the physicochemical properties, antigenic sites, and three-dimensional structure of the mutated protein. The results revealed that there were 12 and 51 polymorphic loci (single-nucleotide polymorphisms, or SNPs) found in the CH1 and CH2 sequences, respectively, with most loci located in the GC-rich intron 2, while others were found in exon 2, intron 3, and exon 3. These SNPs guided sgRNA design by identifying conserved regions in the CH2 gene. The gene editing efficiency for the CH2 region, with this dual CRISPR system, was 40%, with 35% attributed to Type 1 mutation and 5% to Type 2 mutation. In conclusion, CH1 is significantly more conserved than CH2, and the antigenicity of the Fel d1 CH2 gene in domestic cats can be effectively reduced through CRISPR–Cas9 gene editing. Full article

Microcystis aeruginosa is an important species causing cyanobacterial blooms, which can be effectively infected and lysed by cyanophages. Several strategies have been developed by M. aeruginosa to resist cyanophage infections, including the CRISPR-Cas systems. However, detailed information on the CRISPR-Cas systems in M. aeruginosa is rare. In the present study, the CRISPR-Cas systems of M. aeruginosa FACHB-524 were analyzed by genome re-sequencing, which showed that there are two type I (Cluster 1, I-B1; Cluster 2, I-D) and three type III-B (Cluster 3/4/5) CRISPR-Cas systems in the cyanobacteria. Further comparison revealed that spacer sequences of two type III-B systems targeted several genes of the cyanophage MaMV (M. aeruginosa myovirus) strains. One of the type III systems (Cluster 4) was then cloned and expressed in Escherichia coli BL21 (DE3). Protein purification and mass spectrometry identification revealed that a Cmr-crRNA effector complex formed in the E. coli. Subsequently, T4 phage (T4) was used to infect the E. coli, expressing the Cmr-crRNA complex with or without accessory proteins. The results showed that the Cmr-crRNA effector complex exhibited anti-phage activity and the accessory protein Csx1 enhanced the immune activity of the complex. Collectively, our results comprehensively demonstrate the CRISPR systems encoded by a strain of M. aeruginosa, and for the first time, one of the CRISPR systems was constructed into E. coli, providing a foundation for further in-depth analysis of cyanobacterial CRISPR systems. Full article

Skeletal muscle plays a pivotal role in physical activity, protein storage and energy utilization. Skeletal muscle wasting due to immobilization, aging, muscular dystrophy and cancer cachexia has negative impacts on the quality of life. The deletion of myostatin, a growth and differentiation factor-8 (GDF-8) augments muscle mass through hyperplasia and hypertrophy of muscle fibers. The present study examines the impact of myostatin deletion using CRISPR/Cas9 editing on the myogenic differentiation (MD) of C2C12 muscle stem cells. A total of five myostatin loci were targeted using guided RNAs that had been previously cloned into a vector. The clones were transfected in C2C12 cells via electroporation. The cell viability and MD of myostatin-edited clones (Mstn^−/−) were compared with C2C12 (Mstn^+/+) using a series of assays, including MTT, sulforhodamine B, immunocytochemistry, morphometric analysis and RT-qPCR. The clones sequenced showed evidence of nucleotides deletion in Mstn^−/− cells. Mstn^−/− cells demonstrated a normal physiological performance and lack of cytotoxicity. Myostatin depletion promoted the myogenic commitment as evidenced by upregulated MyoD and myogenin expression. The number of MyoD-positive cells was increased in the differentiated Mstn^−/− clones. The Mstn^−/− editing upregulates both mTOR and MyH expression, as well as increasing the size of myotubes. The differentiation of Mstn^−/− cells upregulates ActRIIb; in contrast, it downregulates decorin expression. The data provide evidence of successful CRISPR/Cas9-mediated myostatin deletion. In addition, targeting myostatin could be a beneficial therapeutic strategy to promote MD and to restore muscle loss. In conclusion, the data suggest that myostatin editing using CRISPR/Cas9 could be a potential therapeutic manipulation to improve the regenerative capacity of muscle stem cells before in vivo application. Full article

Filamin C (FLNC) is a structural protein of muscle fibers. Mutations in the FLNC gene are known to cause myopathies and cardiomyopathies in humans. Here we report the generation by a CRISPR/Cas9 editing system injected into zygote pronuclei of two mouse strains carrying filamin C mutations—one of them (AGA) has a deletion of three nucleotides at position c.7418_7420, causing E>>D substitution and N deletion at positions 2472 and 2473, respectively. The other strain carries a deletion of GA nucleotides at position c.7419_7420, leading to a frameshift and a premature stop codon. Homozygous animals (Flnc^AGA/AGA and Flnc^GA/GA) were embryonically lethal. We determined that Flnc^GA/GA embryos died prior to the E12.5 stage and illustrated delayed development after the E9.5 stage. We performed histological analysis of heart tissue and skeletal muscles of heterozygous strains carrying mutations in different combinations (Flnc^GA/wt, Flnc^AGA/wt, and Flnc^GA/AGA). By performing physiological tests (grip strength and endurance tests), we have shown that heterozygous animals of both strains (Flnc^GA/wt, Flnc^AGA/wt) are functionally indistinguishable from wild-type animals. Interestingly, compound heterozygous mice (Flnc^GA/AGA) are viable, develop normally, reach puberty and it was verified by ECG and Eco-CG that their cardiac muscle is functionally normal. Intriguingly, Flnc^GA/AGA mice demonstrated better results in the grip strength physiological test in comparison to WT animals. We also propose a structural model that explains the complementary interaction of two mutant variants of filamin C. Full article

DNA-free genome editing by the direct delivery of CRISPR-associated nucleases has emerged as a promising technology due to its precision and reduced risk of off-target effects. However, existing purification protocols for native Cas proteins require the use of complex instrumentation, which limits their application. Here, we present a simplified protocol for the purification of native Cas9, Cas12RR and dCas9-VP64 nucleases optimized for DNA-free genome editing. Our approach leverages a streamlined affinity and ion exchange chromatography coupled with minimal downstream processing, ensuring a good yield and activity of the purified proteins. The in vitro analysis of the purified ribonucleoprotein complex demonstrated a good efficiency of DNA target cleavage. This simplified protocol increases the opportunity to adopt CRISPR technology, and enables broader access to DNA-free genome editing tools also for laboratories that are not specifically equipped for protein purification. Full article

CRISPR/Cas9-mediated homology-directed repair (HDR) is a powerful tool for precise genome editing in plants, but its efficiency remains low, particularly for targeted amino acid substitutions or gene knock-ins. Successful HDR requires the simultaneous presence of Cas9, guide RNA, and a repair template (RT) in the same cell nucleus. Among these, the timely availability of the RT at the double-strand break (DSB) site is a critical bottleneck. To address this, we developed a sequential transformation strategy incorporating a deconstructed wheat dwarf virus (dWDV)-based autonomously replicating delivery system, effectively simplifying the process into a two-component system. Using this approach, we successfully achieved the targeted editing of the OsEPSPS gene in rice with a 10 percent HDR efficiency, generating three lines (TIPS1, TIPS2, and TIPS3) with amino acid substitutions (T172I and P177S) in the native EPSPS protein. The modifications were confirmed through Sanger sequencing and restriction digestion assays, and the edited lines showed no yield penalties compared to wild-type plants. This study demonstrates the utility of viral replicons in delivering gene-editing tools for precise genome modification, offering a promising approach for efficient HDR in crop improvement programs. Full article

In previous studies, titanium peroxide nanoparticles (PAA-TiOx NPs) with surfaces functionalized using polyacrylic acid (PAA) and hydrogen peroxide (H₂O₂) demonstrated a synergistic effect when combined with X-ray irradiation. The combination generated H₂O₂ and reactive oxygen species (ROS) that enhanced the irradiation efficacy. In the present study, we examined the relationship between catalase and PAA-TiOx NPs sensitization to X-ray radiation because catalase is the primary antioxidant enzyme that converts H₂O₂ to water and oxygen. Catalase-knockout PANC-1 (dCAT) cells were generated using the CRISPR/Cas9 system, which was confirmed by the suppression of catalase expression in mRNA and protein levels that resulted in an 81.7% decrease in catalase activity compared with levels in wild-type cells. Catalase deficiency was found to increase the production of ROS, particularly in hypoxia. Also, the combination of PAA-TiOx NPs and X-ray 5 Gy resulted in a 7-fold decrease in the survival fraction (SF; p < 0.01) of dCAT cells compared with rates documented in wild-type cells. Interestingly, the combination treatment with X-ray 3 Gy in dCAT cells resulted in an SF similar to that observed in wild-type cells treated with the same combination but at a higher radiation dose (5 Gy). These results suggest that a strategy of catalase inhibition could be used to establish an advanced combination treatment of PAA-TiOx NPs and X-ray irradiation for pancreatic cancer cells. Full article

Breast cancer (BC) is a widespread malignancy that affects the lives of millions of women each year, and its resulting financial and healthcare hardships cannot be overstated. These issues, in combination with side effects and obstacles associated with the current standard of care, generate considerable interest in new potential targets for treatment as well as means for BC prevention. One potential preventive compound is Withaferin A (WFA), a traditional medicinal compound found in winter cherries. WFA has shown promise as an anticancer agent and is thought to act primarily through its effects on the epigenome, including, in particular, the methylome. However, the relative importance of specific genes’ methylation states to WFA function remains unclear. To address this, we utilized human BC cell lines in combination with CRISPR-dCas9 fused to DNA methylation modifiers (i.e., epigenetic editors) to elucidate the importance of specific genes’ promoter methylation states to WFA function and cancer cell viability. We found that targeted demethylation of promoters of the tumor suppressors p21 and p53 within MDA-MB-231/MCF7 cells resulted in around 1.7×/1.5× and 1.2×/1.3× increases in expression, respectively. Targeted methylation of the promoter of the oncogene CCND1 within MDA-MB-231/MCF7 cells resulted in 0.5×/0.8× decreases in gene expression. These changes to p21, p53, and CCND1 were also associated with decreases in cell viability of around 25%/50%, 5%/35%, and 12%/16%, respectively, for MDA-MB-231/MCF7 cells. When given in combination with WFA in both p53 mutant and wild type cells, we discovered that targeted methylation of the p21 promoter was able to modulate the anticancer effects of WFA, while targeted methylation or demethylation of the promoters of p53 and CCND1 had no significant effect on viability decreases from WFA treatment. Taken together, these results indicate that p21, p53, and CCND1 may be important targets for future in vivo studies that may lead to epigenetic editing therapies and that WFA may have utility in the prevention of BC through its effect on p21 promoter methylation independent of p53 function. Full article

Invariant Natural Killer T (iNKT) cells are a unique subset of T cells that bridge innate and adaptive immunity, displaying potent anti-tumor properties through cytokine secretion, direct cytotoxicity, and recruitment of immune effector cells such as CD8⁺ T cells and NK cells. Despite their therapeutic potential, the immunosuppressive tumor microenvironment (TME), characterized by regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs), limits iNKT cell efficacy. Patient-derived organoid (PDO) platforms provide an innovative model for dissecting these complex interactions and evaluating strategies to reinvigorate iNKT cell functionality within the TME. PDOs closely mimic the genetic, phenotypic, and structural characteristics of primary tumors, enabling the study of tumor–immune dynamics. Integrating iNKT cells into PDOs offers a robust platform for investigating CD1d-mediated interactions, Th1-biased immune responses driven by glycolipid analogs like α-GalCer, and combination therapies such as immune checkpoint inhibitors. Additionally, PDO systems can assess the effects of metabolic modulation, including reducing lactic acid accumulation or targeting glutamine pathways, on enhancing iNKT cell activity. Emerging innovations, such as organoid-on-a-chip systems, CRISPR-Cas9 gene editing, and multi-omics approaches, further expand the potential of PDO–iNKT platforms for personalized immunotherapy research. Although the application of iNKT cells in PDOs is still undeveloped, these systems hold immense promise for bridging preclinical studies and clinical translation. By addressing the challenges of the TME and optimizing therapeutic strategies, PDO–iNKT platforms offer a transformative avenue for advancing cancer immunotherapy and personalized medicine. Full article