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Applications of CRISPR-Cas9 in Alzheimer's Disease and Related Disorders.
Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease represent some of the most prevalent neurodegenerative disorders afflicting millions of people worldwide. Unfortunately, there is a lack of efficacious treatments to cure or stop the progression of these disorders. While the causes of such a lack of therapies can be attributed to various reasons, the disappointing results of recent clinical trials suggest the need for novel and innovative approaches. Since its discovery, there has been a growing excitement around the potential for CRISPR-Cas9 mediated gene editing to identify novel mechanistic insights into disease pathogenesis and to mediate accurate gene therapy. To this end, the literature is rich with experiments aimed at generating novel models of these disorders and offering proof-of-concept studies in preclinical animal models validating the great potential and versatility of this gene-editing system. In this review, we provide an overview of how the CRISPR-Cas9 systems have been used in these neurodegenerative disorders.
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Limit on lab-grown human embryos dropped by stem-cell body.
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CRISPR/Cas9, the Powerful New Genome-Editing Tool for Putative Therapeutics in Obesity.
The molecular technology known as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is revolutionizing the field of medical research and deepening our understanding of numerous biological processes. The attraction of CRISPR/Cas9 lies in its ability to efficiently edit DNA or modulate gene expression in living eukaryotic cells and organisms, a technology that was once considered either too expensive or scientifically risky. CRISPR/Cas9 has been successfully applied in agriculture to develop the next generation of disease-resistant plants. Now, the capability of gene editing has been translated to the biomedical area, focusing on the future of medicine faced with drug-resistant microbes by selectively targeting genes involved in antibiotic resistance, for example, or finding the ultimate strategy for cancer or HIV. In this regard, it was recently demonstrated that an injection of cancer-fighting CRISPR-modified white blood cells in a patient suffering from metastatic lung cancer could lead to promising results. Researchers and bioethicists are debating questions about the regulation of CRISPR/Cas9 that must be addressed. While legal challenges surround the use of this technique for genetically modifying cell lines in humans, we review the basic understanding of CRISPR/Cas9 and discuss how this technology could represent a candidate for treatment of non-communicable diseases in nutrition, such as obesity.
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Optimization of RNP-CRISPR for high-efficiency gene editing in mouse intestinal organoids.
Mouse intestinal organoids are ideal models for investigating intestinal development and diseases. The full potential of these models hinges on the ability to precisely engineer their genome, yet traditional methods for CRISPR-based editing in 3D cultures often surfer from low efficiency, high cytotoxicity, and inconsistent post-editing differentiation, which limits their applications. Here, we developed an electroporation approach mediated by ribonucleoprotein (RNP)-CRISPR that achieves over 90% gene editing efficiency in mouse intestinal organoids. Using this optimized method, we generated APC-knockout organoids that exhibit Wnt pathway hyperactivation, demonstrated by R-spondin1-independent growth, increased nuclear β-catenin, and enhanced proliferation. Our method addresses a critical technical gap in murine organoid research, offering a scalable platform for intestinal disease modeling.
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A CASE toolkit for easy and efficient multiplex transgene-free gene editing.
An integrated transgene-free multiplex gene-editing toolkit based on the Transgene Killer CRISPR technology greatly saves labor, time, and cost.
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[A universal counter-selection strategy based on replacement of sgRNA expression cassettes targeting multi-copy genes].
Selection markers are essential tools in gene editing, the utility of such systems is inherently constrained by species-specific limitations, governed by divergent host genetic backgrounds and metabolic compatibility. To address this limitation, we leveraged the CRISPR/Cas9 system to develop a universal counter-selection tool. We designed and introduced an sgRNA expression cassettes as counter-selection markers, which directs the Cas9 protein to target and cleave genomic DNA, allowing for the selection of the strains where the sgRNA expression cassette has been replaced. Optimized to target multiple copy sites with sgRNA, this system significantly enhances cell lethality, boosting counter-selection efficiency to over 85.00%. This counter-selection tool is not limited to single strains and is suitable for various scenarios, including multi-copy plasmid assembly and plasmid editing, demonstrating broad application potential.
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Integrase-Deficient Lentiviral Vector as a Platform for Efficient CRISPR/Cas9-Mediated Gene Editing for Mucopolysaccharidosis IVA.
Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder causing systemic skeletal dysplasia due to a deficiency of N-acetyl-galactosamine-6-sulfate sulfatase (GALNS) enzyme activity, leading to the impaired degradation and accumulation of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate. While treatments such as enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are available, they have significant limitations regarding efficacy in skeletal tissues and long-term safety, highlighting the need for more effective therapies. We evaluated a novel gene therapy approach using a dual Integrase-deficient lentiviral vector (IDLV) to deliver an expression cassette that includes human GALNS cDNA and Cas9 sgRNA, targeting the upstream region of the mouse Galns initial codon. This approach leverages the endogenous promoter to drive transgene expression. We assessed in vitro transduction, editing, and functional correction in NIH3T3 and MPS IVA mouse fibroblasts. In vivo efficacy was successfully evaluated via the facial vein injection in MPS IVA newborn mice. In vitro, this IDLV platform demonstrated supraphysiological GALNS activity in cell lysate, resulting in the normalization of KS levels. In vivo direct IDLV platform in newborn MPS IVA mice led to sustained plasma GALNS activity, reduced plasma KS, and favorable biodistribution. Partial correction of heart and bone pathology was observed, with no vector toxicity and minimal antibody responses. This dual IDLV-CRISPR/Cas9 approach effectively mediated targeted GALNS knock-in, yielding sustained enzyme activity, reduced KS storage, and partial pathological amelioration in MPS IVA mice. In conclusion, IDLVs represent an efficient, safe platform for delivering the CRISPR/Cas9 gene editing system for MPS IVA.
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Single-Step Genome Editing of Small Ruminant Embryos by Electroporation.
We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos.
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Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes.
The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.
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Alpha-1 Antitrypsin Deficiency.
Alpha-1 antitrypsin (AAT) deficiency is a common monogenic disorder in which there is a strong founder effect of a single missense mutation in SERPINA1, the gene encoding this major circulating serum anti-protease that is normally expressed primarily in hepatocytes. These features make AAT deficiency particularly attractive as a target for therapeutic gene editing using a wide variety of approaches.
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