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CRISPR/Cas9-mediated gene editing on Sox2ot promoter leads to its truncated expression and does not influence neural tube closure and embryonic development in mice.
Sox2 overlapping transcript (Sox2ot) is a long non-coding RNA (lncRNA), which harbors one of the major regulators of pluripotency, the Sox2 gene, in its intronic region. Sox2ot is primarily expressed in the developing neuroepithelium. However, its role in neural tube closure and embryonic development remains unclear. To investigate if Sox2ot is required for neural tube closure and embryonic development, Sox2ot promoter was deleted by CRISPR-Cas9 genome editing technology to prevent Sox2ot gene expression in mice. We designed 9 guide RNAs to specifically target the Sox2ot promoter and 3 gRNAs induced gene editing on the promoter of the Sox2ot gene in cells transfected with Cas9 mRNA and gRNAs. Then, these gRNAs and Cas9 mRNA were injected into mouse zygotes and implanted into pseudopregnant mice. A Sox2ot promoter-deleted mouse line was identified with complete deletion of promoter as well as deletion of exon 1 and exon 2. Sox2ot transcript was truncated with a lack of exon 1 and exon 2 in Sox2ot promoter-deleted mice. Furthermore, neural tube closure and embryonic development were checked at E9.5, E10.5, E14.5, E17.5 and after-birth (P2) and we did not find any failure of neural tube closure and aberrant embryonic development in Sox2ot promoter-deleted mice. Thus, our study demonstrated that CRISPR-Cas9 gene editing in Sox2ot promoter leads to its truncated expression and does not influence neural tube closure and embryonic development.
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Generation of mutant pigs by lipofection-mediated genome editing in embryos.
The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.
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Targeted genome editing in the rare actinomycete Actinoplanes sp. SE50/110 by using the CRISPR/Cas9 System.
The application of genome editing technologies, like CRISPR/Cas9 for industrially relevant microorganisms, is becoming increasingly important. Compared to other methods of genetic engineering the decisive factor is that CRISPR/Cas9 is relatively easy to apply and thus time and effort can be significantly reduced in organisms, which are otherwise genetically difficult to access. Because of its many advantages and opportunities, we adopted the CRISPR/Cas9 technology for Actinoplanes sp. SE50/110, the producer of the diabetes type II drug acarbose. The functionality of genome editing was successfully shown by the scarless and antibiotic marker-free deletion of the gene encoding the tyrosinase MelC, which catalyzes the formation of the dark pigment eumelanin in the wild type strain. The generated ΔmelC2 mutant of Actinoplanes sp. SE50/110 no longer produces this pigment and therefore the supernatant does not darken. Furthermore, it was shown that the plasmid containing the gene for the Cas9 protein was removed by increasing the temperature due to its temperature-sensitive replication. The precision of the intended mutation was proven and possible off-target effects caused by the genome editing system were ruled out by genome sequencing of several mutants.
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Lipoplex as a useful tool for resensitization of methicillin-resistant Staphylococcus aureus to erythromycin via CRISPR-Cas technology.
The global escalation of antimicrobial resistance (AMR) necessitates innovative therapeutic interventions. Methicillin-resistant Staphylococcus aureus (MRSA) remains a primary clinical threat due to its extensive resistance profile. In this study, CRISPR-Cas9 technology was employed to target the ermA gene, which is responsible for erythromycin resistance in clinical MRSA isolates. A cationic liposome (Lip) composed of cholesterol, DOTAP, and DOPE was developed to deliver the pCasSA plasmid. Particle characterization revealed stable liposomes (Lips) (<200 nm, PDI < 0.3, ZP>+30 mV). pCasSA was loaded into the Lip, to form a lipoplex (Lpx), a complex of Lip and pCasSA, via electrostatic interactions. Cytotoxicity studies using mouse fibroblast L929 cells revealed that cell viabilities remained at approximately 93% and 83% following treatment with the highest concentration of Lip and Lpx formulations, respectively. Successful gene editing was confirmed via Sanger sequencing and RT-qPCR, showing a 75% reduction in ermA expression when Lpx wascombined with sonoporation. Phenotypic assays demonstrated a significant restoration of susceptibility. The minimum inhibitory concentration (MIC) of erythromycin decreased 8-fold (from 8 mg/L to 1 mg/L), and inhibition zones increased 2.5-fold (from 12 mm to 30 mm). These findings suggest that Lpx-mediated CRISPR-Cas9 delivery is a highly efficient strategy for antibiotic resensitization, potentially restoring the clinical utility of existing antimicrobials.
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The Role of CRISPR and Its Therapeutic Applications in Glioblastoma.
Glioblastoma multiforme (GBM) remains the most aggressive and treatment-refractory form of primary brain tumor in adults, characterized by rapid proliferation, intratumoral heterogeneity and resistance to current therapies. Despite therapeutic advancements in surgical resection, radiotherapy and chemotherapy, clinical outcomes remain poor, underscoring the need for innovative molecular strategies. This review examines the therapeutic potential of CRISPR/Cas9 genome-editing technologies in GBM, highlighting their ability to model, dissect and potentially correct the genetic alterations that drive GBM tumorigenesis. Key molecular targets, such as EGFR, PTEN, TP53, NF1 and PIK3CA, are discussed within the context of GBM's mutational and signaling landscape. We further outline emerging CRISPR applications in preclinical models, the current status of CRISPR-based clinical trials and the major barriers hindering translation, including off-target effects, immunogenicity and the challenge of delivering gene-editing systems across the blood-brain barrier. Particular emphasis is placed on delivery technologies, viral and non-viral vectors, including lipid nanoparticles, polymeric systems, inorganic nanocarriers and DNA nanostructures, which are rapidly evolving to improve precision, safety and CNS penetrance. Collectively, this review highlights CRISPR/Cas9 as a powerful tool whose integration with molecular neuro-oncology and precision medicine may ultimately shift GBM treatment toward more personalized and durable therapeutic interventions.
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Cationic lipid nanoparticle-mediated delivery of a Cas9/crRNA ribonucleoprotein complex for transgene-free editing of the citrus plant genome.
A lipofectamine-mediated transfection protocol for DNA-free genome editing of citrus protoplast cells using a Cas9/gRNA ribonucleoprotein (RNP) complex resulted in the production of transgene free genome edited citrus.
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Generation of Genomic Deletions (of Rig-I GENE) in Goat Primary Cell Culture Using CRISPR/CAS9 Method.
CRISPR/Cas9 system is a natural immune system in prokaryotes protecting them from infectious viral or plasmid DNA invading the cells. This RNA-guided system can act as powerful tool for introducing genomic alterations in eukaryotic cells with high efficiency. In the present study, Rig-Igene is taken as model gene to study the efficiency of CRISPR/Cas9 system induced gene deletion in primary fibroblast cell culture. Rig-I(retinoic acid-inducible gene-1) is involved in regulating immune response in mammals. In this study, we optimized the CRISPR/Cas9 method for knocking out Rig-Igene in Goat primary fibroblasts by using a NHEJ pathway. Cells were screened for inactivation of the Rig-Igene and two positive clones were found out of thirty colonies screened. Thus, cells containing Rig-Igene inactivation could be achieved by CRISPR/Cas9 in goat fibroblast cells.
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Debate on Germline Gene Editing.
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Effective Genome Editing Using CRISPR-Cas9 Nanoflowers.
CRISPR-Cas9 as a powerful gene-editing tool has tremendous potential for the treatment of genetic diseases. Herein, a new mesoporous nanoflower (NF)-like delivery nanoplatform termed Cas9-NF is reported by crosslinking Cas9 and polymeric micelles that enables efficient intracellular delivery and controlled release of Cas9 in response to reductive microenvironment in tumor cells. The flower morphology is flexibly tunable by the protein concentration and different types of crosslinkers. Cas9 protein, embedded between polymeric micelles and protected by Cas9-NF, remains stable even under extreme pH conditions. Responsive cleavage of crosslinkers in tumor cells, leads to the traceless release of Cas9 for efficient gene knockout in nucleus. This crosslinked nanoparticle exhibits excellent capability of downregulating oncogene expression and inhibiting tumor growth in a murine tumor model. Taken together, these findings pave a new pathway toward the application of the protein-micelle crosslinked nanoflower for protein delivery, which warrants further investigations for gene regulation and cancer treatment.
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Applications of CRISPR technology in cellular immunotherapy.
CRISPR technology has transformed multiple fields, including cancer and immunology. CRISPR-based gene editing and screening empowers direct genomic manipulation of immune cells, opening doors to unbiased functional genetic screens. These screens aid in the discovery of novel factors that regulate and reprogram immune responses, offering novel drug targets. The engineering of immune cells using CRISPR has sparked a transformation in the cellular immunotherapy field, resulting in a multitude of ongoing clinical trials. In this review, we discuss the development and applications of CRISPR and related gene editing technologies in immune cells, focusing on functional genomics screening, gene editing-based cell therapies, as well as future directions in this rapidly advancing field.
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