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Designer nuclease-mediated gene correction via homology-directed repair in an in vitro model of canine hemophilia B.
BACKGROUND: Gene correction at specific target loci provides a powerful strategy for overcoming genetic diseases. In the present study, we aimed to use an in vitro model for canine hemophilia B containing a single point mutation in the catalytic domain of the canine coagulation factor IX (cFIX) gene. To correct the defective gene via homology-directed repair (HDR), we designed transcription-activator like effector nucleases and clustered regularly interspaced short palindromic repeats including Cas9 (CRISPR/Cas9) for introduction of double-strand breaks at the mutation site. METHODS: To generate a stable cell line containing the mutated cFIX locus, a 2-kb genomic DNA fragment derived from a hemophilia B dog was amplified and integrated utilizing the phiC31 integrase system. Designer nucleases were assembled and cloned into vectors for constitutive and inducible expression. To detect mutations, insertions and deletions, and HDR events after nuclease treatment T7E1 assays, an amplification-refractory mutation system-quantitative polymerase chain reaction and pyrosequencing were performed. RESULTS: To perform HDR correction experiments, we established a cell line carrying the mutated cFIX locus. In HDR approaches we either explored a wild-type or an optimized cFIX sequence and we found that our modified HDR cassette showed higher gene correction efficiencies of up to 6.4%. Furthermore, we compared inducible and constitutive designer nuclease expression systems and found that the inducible system resulted in comparable HDR efficiencies. CONCLUSIONS: In conclusion, the present study demonstrates the potential of this strategy for gene therapeutic approaches in vitro and in a canine model for hemophilia B.
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Succinic acid production from softwood with genome-edited Corynebacterium glutamicum using the CRISPR-Cpf1 system.
Corynebacterium glutamicum is a useful microbe that can be used for producing succinic acid under anaerobic conditions. In this study, we generated a knock-out mutant of the lactate dehydrogenase 1 gene (ΔldhA-6) and co-expressed the succinic acid transporter (Psod:SucE- ΔldhA) using the CRISPR-Cpf1 genome editing system. The highly efficient HPAC (hydrogen peroxide and acetic acid) pretreatment method was employed for the enzymatic hydrolysis of softwood (Pinus densiflora) and subsequently utilized for production of succinic acid. Upon evaluating a 1%-5% hydrolysate concentration range, optimal succinic acid production with the ΔldhA mutant was achieved at a 4% hydrolysate concentration. This resulted in 14.82 g L-1 succinic acid production over 6 h. No production of acetic acid and lactic acid was detected during the fermentation. The co-expression transformant, [Psod:SucE-ΔldhA] produced 17.70 g L-1 succinic acid in 6 h. In the fed-batch system, 39.67 g L-1 succinic acid was produced over 48 h. During the fermentation, the strain consumed 100% and 73% of glucose and xylose, respectively. The yield of succinic acid from the sugars consumed was approximately 0.77 g succinic acid/g sugars. These results indicate that the production of succinic acid from softwood holds potential applications in alternative biochemical processes.
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Corneal xenotransplantation: Where are we standing?
The search for alternatives to allotransplants is driven by the shortage of corneal donors and is demanding because of the limitations of the alternatives. Indeed, current progress in genetically engineered (GE) pigs, the introduction of gene-editing technology by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, and advanced immunosuppressants have made xenotransplantation a possible option for a human trial. Porcine corneal xenotransplantation is considered applicable because the eye is regarded as an immune-privileged site. Furthermore, recent non-human primate studies have shown long-term survival of porcine xenotransplants in keratoplasty. Herein, corneal immune privilege is briefly introduced, and xenogeneic reactions are compared with allogeneic reactions in corneal transplantation. This review describes the current knowledge on special issues of xenotransplantation, xenogeneic rejection mechanisms, current immunosuppressive regimens of corneal xenotransplantation, preclinical efficacy and safety data of corneal xenotransplantation, and updates of the regulatory framework to conduct a clinical trial on corneal xenotransplantation. We also discuss barriers that might prevent xenotransplantation from becoming common practice, such as ethical dilemmas, public concerns on xenotransplantation, and the possible risk of xenozoonosis. Given that the legal definition of decellularized porcine cornea (DPC) lies somewhere between a medical device and a xenotransplant, the preclinical efficacy and clinical trial data using DPC are included. The review finally provides perspectives on the current standpoint of corneal xenotransplantation in the fields of regenerative medicine.
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The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones.
Recent advances in genome editing systems such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) have facilitated genomic modification in mammalian cells. However, most systems employ transient treatment with selective drugs such as puromycin to obtain the desired genome-edited cells, which often allows some untransfected cells to survive and decreases the efficiency of generating genome-edited cells. Here, we developed a novel targeted toxin-based drug-free selection system for the enrichment of genome-edited cells. Cells were transfected with three expression vectors, each of which carries a guide RNA (gRNA), humanized Cas9 (hCas9) gene, or Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene. Once EndoGalC is expressed in a cell, it digests the cell-surface α-Gal epitope, which is specifically recognized by BS-I-B₄ lectin (IB4). Three days after transfection, these cells were treated with cytotoxin saporin-conjugated IB4 (IB4SAP) for 30 min at 37 °C prior to cultivation in a normal medium. Untransfected cells and those weakly expressing EndoGalC will die due to the internalization of saporin. Cells transiently expressing EndoGalC strongly survive, and some of these surviving clones are expected to be genome-edited bi-allelic knockout (KO) clones due to their strong co-expression of gRNA and hCas9. When porcine α-1,3-galactosyltransferase gene, which can synthesize the α-Gal epitope, was attempted to be knocked out, 16.7% and 36.7% of the surviving clones were bi-allelic and mono-allelic knockout (KO) cells, respectively, which was in contrast to the isolation of clones in the absence of IB4SAP treatment. Namely, 0% and 13.3% of the resulting clones were bi-allelic and mono-allelic KO cells, respectively. A similar tendency was seen when other target genes such as DiGeorge syndrome critical region gene 2 and transforming growth factor-β receptor type 1 gene were targeted to be knocked out. Our results indicate that a combination of the CRISPR/Cas9 system and targeted toxin technology using IB4SAP allows efficient enrichment of genome-edited clones, particularly bi-allelic KO clones.
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Muscle-specific gene editing improves molecular and phenotypic defects in a mouse model of myotonic dystrophy type 1.
BACKGROUND: Myotonic dystrophy type 1 (DM1) is a genetic multisystemic disease, characterised by pleiotropic symptoms that exhibit notable variability in severity, nature and age of onset. The genetic cause of DM1 is the expansion of unstable CTG-repeats in the 3' untranslated region (UTR) of the DMPK gene, resulting in the accumulation of toxic CUG-transcripts that sequester RNA-binding proteins and form nuclear foci in DM1 affected tissues and, consequently, alter various cellular processes. Therapeutic gene editing for treatment of monogenic diseases is a powerful technology that could in principle remove definitively the disease-causing genetic defect. The precision and efficiency of the molecular mechanisms are still under investigation in view of a possible use in clinical practice. METHODS: Here, we describe the application of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) strategy to remove the CTG-expansion in the DMPK gene in a mouse model carrying the human transgene from a DM1 patient. To optimise the editing efficiency in vivo, we identified new tools that allowed to improve the expression levels and the activity of the CRISPR/Cas9 machinery. Newly designed guide RNA pairs were tested in DM1-patient derived cells before in vivo application. Edited cells were analysed to assess the occurrence of off-target and the accuracy of on-target genomic events. Gene editing-dependent and -independent mechanisms leading to decreased accumulation of the mutated DMPK transcripts were also evaluated. RESULTS AND CONCLUSION: Systemic delivery of CRISPR/Cas9 components in DM1 mice, through myotropic adeno-associated viral vectors, led to significant improvement of molecular alterations in the heart and skeletal muscle. Importantly, a persistent increase of body weight, improvement of muscle strength and body composition parameters were observed in treated animals. Accurate evaluation of CRISPR/Cas9-mediated-phenotypic recovery in vivo is a crucial preclinical step for the development of a gene therapy for DM1 patients. KEY POINTS: In vivo application of a therapeutic gene editing strategy for permanent deletion of the pathogenetic CTG-repeat amplification in the DMPK gene that causes myotonic dystrophy type 1. Following treatment, diseased mice show a significant improvement of both molecular and phenotypic defects.
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CRISPR applications in ophthalmologic genome surgery.
PURPOSE OF REVIEW: The present review seeks to summarize and discuss the application of clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) for genome editing, also called genome surgery, in the field of ophthalmology. RECENT FINDINGS: Precision medicine is an emerging approach for disease treatment and prevention that takes into account the variability of an individual's genetic sequence. Various groups have used CRISPR-Cas genome editing to make significant progress in mammalian preclinical models of eye disease, the basic science of eye development in zebrafish, the in vivo modification of ocular tissue, and the correction of stem cells with therapeutic applications. In addition, investigators have creatively used the targeted mutagenic potential of CRISPR-Cas systems to target pathogenic alleles in vitro. SUMMARY: Over the past year, CRISPR-Cas genome editing has been used to correct pathogenic mutations in vivo and in transplantable stem cells. Although off-target mutagenesis remains a concern, improvement in CRISPR-Cas technology and careful screening for undesired mutations will likely lead to clinical eye therapeutics employing CRISPR-Cas systems in the near future.
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CRISPR-Enabled functional genomics in hPSCs-derived neural models for autism spectrum disorder.
Autism Spectrum Disorder (ASD) is a genetically heterogeneous neurodevelopmental condition in which hundreds of individually rare risk variants converge on a small number of shared biological pathways, including synaptic scaffolding, chromatin remodeling, excitation-inhibition balance, and cellular energy metabolism. Translating this genetic heterogeneity into mechanistic insight requires experimental systems capable of interrogating individual gene functions in human-relevant neural contexts at scale. CRISPR-enabled functional genomics in human pluripotent stem cell (hPSC)-derived neural models, spanning neural progenitors, cortical and inhibitory neurons, astrocytes, microglia, and brain organoids, provides precisely this capability. By integrating pooled perturbation screens with multimodal readouts including single-cell and spatial transcriptomics, chromatin accessibility profiling, proximity labeling proteomics, multi-electrode array electrophysiology, and metabolic flux analysis, these platforms enable systematic, causal mapping of ASD gene function at system resolution. Early applications have already revealed convergent mechanisms: BAF complex disruption expands the ventral progenitor pool and biases its fate toward oligodendrocyte and interneuron lineages; ADNP loss impairs microglial synaptic pruning through altered endocytic trafficking; and mTOR pathway dysregulation in PTEN- and TSC2-perturbed models links genetic risk directly to metabolic and mitochondrial dysfunction. Computational frameworks including MIMOSCA and SCEPTRE enable causal network reconstruction and pseudotime inference from these datasets, moving the field from gene lists toward pathway-level models of ASD pathobiology. Translational applications leverage isogenic iPSC panels and variant-level base and prime editing to stratify ASD variants by functional impact, informing gene therapy design for haploinsufficient targets such as CHD8 and SCN2A via AAV or antisense oligonucleotide delivery. Remaining challenges, including model developmental immaturity, batch variability, and the difficulty of modeling polygenic risk, are addressed by a roadmap integrating spatial perturbomics, AI-driven causal inference, and population-scale standardized biobanks. This review synthesizes the current state of CRISPR-based functional genomics in human stem cell neural models as a coherent experimental framework for converting ASD genetic associations into mechanistic understanding and therapeutic opportunity.
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Insights into the mechanisms underlying the inactivation of HIV-1 proviruses by CRISPR/Cas.
DNA editing using CRISPR/Cas has emerged as a potential treatment for diseases caused by pathogenic human DNA viruses. One potential target is HIV-1, which replicates via a chromosomally integrated DNA provirus. While CRISPR/Cas can protect T cells from de novo HIV-1 infection, HIV-1 frequently becomes resistant due to mutations in the chosen single guide RNA (sgRNA) target site. To address this problem, we asked whether an sgRNA targeted to a conserved, functionally critical HIV-1 sequence might prevent the selection of escape mutants. We report that two sgRNAs specific for the HIV-1 transactivation response (TAR) element produce opposite results: the TAR2 sgRNA rapidly selects for mutants that retain TAR function, but are no longer inhibited by Cas9, while the TAR1 sgRNA fails to select any replication competent TAR mutants, most probably because it is targeted to a region of TAR that is disrupted by even minor mutations.
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Clustered Regularly Interspaced Short Palindromic Repeats Genome Editing for Cardiovascular Disease: The Future Is Here.
Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has expanded from experimental biology to early clinical application, raising the possibility of durable therapies for cardiovascular disease. Because many cardiac conditions are monogenic, they provide clear targets for allele-specific correction or modulation. In hypertrophic cardiomyopathy, preclinical research has shown that base editing of pathogenic MYH7 and MYBPC3 mutations can restore sarcomere function; concurrently, RNA-targeting approaches selectively suppress mutant transcripts. Dilated cardiomyopathy is more heterogeneous: TTN truncations cause haploinsufficiency that can be offset by CRISPR activation, while RBM20 and LMNA mutations require precise correction or interference to restore splicing and nuclear stability. Genome editing is also being tested in familial hypercholesterolemia, where inactivation of PCSK9 using lipid nanoparticle-delivered base editors has now advanced to first-in-human trials, achieving sustained LDL-C lowering. Concurrently, efforts targeting ANGPTL3 and APOB highlight the prospect of multigene modulation of lipid metabolism. In arrhythmic syndromes, patient-derived cardiomyocytes edited at SCN5A and KCNQ1 genes have enabled high-fidelity disease models, while in vivo correction of RYR2 in catecholaminergic polymorphic ventricular tachycardia confirms the viability of editing an arrhythmia substrate. In cardiac regeneration, CRISPR activation of developmental transcription factors has enabled direct reprogramming of fibroblasts into cardiomyocyte-like cells within scar tissue. Even with these advances, delivery remains a bottleneck due to immune responses to viral vectors, limitations in the efficiency of lipid nanoparticles in the heart, and the precision required to target cardiomyocytes or conduction cells, all of which slow progress. Future work will depend as much on technical refinement as on navigating ethical, regulatory, and societal concerns.
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CRISPR/Cas9-mediated deletion of adipocyte genes associated with NAFLD alters adipocyte lipid handling and reduces steatosis in hepatocytes in vitro.
Obesity is a major risk factor for the development of nonalcoholic fatty liver disease (NAFLD), and the subcutaneous white adipose tissue (scWAT) is the primary lipid storage depot and regulates lipid fluxes to other organs. Our previous work identified genes upregulated in scWAT of patients with NAFLD: SOCS3, DUSP1, and SIK1. Herein, we knocked down (KD) their expression in human adipose-derived mesenchymal stem cells (hADMSCs) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterized their phenotype. We found that SOCS3, DUSP1, and SIK1 expression in hADMSC-derived adipocytes was not critical for adipogenesis. However, the metabolic characterization of the cells suggested that the genes played important roles in lipid metabolism. Reduction of SIK1 expression significantly increased both de novo lipogenesis (DNL) and palmitate-induced lipogenesis (PIL). Editing out SOCS3 reduced DNL while increasing isoproterenol-induced lipolysis and insulin-induced palmitate accumulation. Conversely, DUSP1 reduced PIL and DNL. Moreover, RNA-sequencing analysis of edited cells showed that these genes not only altered lipid metabolism but also other biological pathways related to inflammatory processes, in the case of DUSP1, extracellular matrix remodeling for SOCS3, or cellular transport for SIK1. Finally, to evaluate a possible adipocyte-hepatocyte axis, human hepatoma HepG2 cells were cocultured with edited hADMSCs-derived adipocytes in the presence of [3H]-palmitate. All HepG2 cells cultured with DUSP1-, SIK1-, or SOCS3-KD adipocytes decreased [3H]-palmitate accumulation compared with control adipocytes. These results support our hypotheses that SOCS3, DUSP1, and SIK1 regulate multiple aspects of adipocyte function, which may play a role in the progression of obesity-associated comorbidities, such as NAFLD.NEW & NOTEWORTHY Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology successfully edited genomic DNA of human adipose-derived mesenchymal stem cells (hADMSC). SOCS3, SIK1, and DUSP1 regulate adipocyte lipid handling. Silencing SOCS3, SIK1, and DUSP1 expression in hADMSC-derived adipocytes reduces hepatocyte lipid storage in vitro.
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