Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1132553 | ENA |
REPOSITORIES: ENA
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Project description:CRISPR-Cas9 delivery by AAV holds promise for gene therapy but faces critical barriers due to its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multi-functional platform customizable for genome-editing, transcriptional regulation, and other previously impracticable AAV-CRISPR-Cas9 applications. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce detectable cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics mRNA-Seq from muscles (9 samples; 3 mice x 3 conditions) and lymph nodes (9 samples; 3 mice x 3 conditions).
2016-07-29 | E-GEOD-84534 | biostudies-arrayexpress
Project description:The clinical translation of CRISPR gene editing is challenged by the lack of delivery systems that are both safe and efficient in therapeutically relevant cell types such as mesenchymal stem cells (MSCs). Non-viral delivery avoids the immunogenicity and genomic integration risks of viral vectors but faces fundamental trade-offs between editing efficiency and cytotoxicity. Here, we present a comprehensive multiomic analysis of four non-viral CRISPR delivery modalities including cell-penetrating peptide- (CPP), lipid-, and polymer-based nanoparticles and electroporation; across mRNA and ribonucleoprotein (RNP) molecular formats. We systematically evaluate each modality, demonstrating that lipid-based delivery achieved the highest editing rates at the cost of genomic instability, interferon pathway activation, and a pro-inflammatory shift in the MSC paracrine activity. Alternatively, CPPs yield moderate editing rates while reducing these unintended side-effects, whereas polymers and electroporation consistently yielded the lowest efficiencies. CRISPR molecular format and delivery method interacted in a stress-dependent manner, with RNP delivery reducing editing rates under high-stress systems while improving them in lower-stress modalities such as CPP and electroporation. These findings establish that editing efficiency alone is an insufficient metric for delivery system selection, and that genomic stability, transcriptomic dysregulation, and inflammatory response must be treated as primary design criteria for CRISPR therapies.
2026-06-01 | GSE333489 | GEO
Project description:Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.
| PRJEB12161 | ENA
Project description:Repair of double-strand DNA breaks generated by site-directed endonucleases, like Cas9, is the hallmark of gene editing based on homology-directed repair (HDR). HDR uses an exogenous DNA template to restore the cleaved DNA sequence and can facilitate specific gene corrections as well as insertion of genes or partial cDNA sequences. For CRISPR/Cas-directed gene editing, co-administration of the Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complex and a DNA template typically involves two different delivery strategies or different types of vehicles. This requires exquisite timing of delivery and may potentially challenge safety and therapeutic applicability. There is a need therefore for technologies that can ferry complete editing tool kits into cells. Here, we demonstrate the use of lentivirus-derived nanoparticles (LVNPs) to transport both RNP complexes and vector RNA, which upon reverse transcription serves as donor for HDR-directed repair. These ‘all-in-one’ LVNPs support targeted gene insertion with reduced off-target effects relative to nucleofection procedures. We show potent editing in the HBB gene in human erythroid progenitor cells as well as HDR-directed editing in hematopoietic stem and progenitor cells. Our findings mark a first step toward using a single virus-derived vehicle for delivering a full HDR gene editing kit.
2025-08-14 | GSE283171 | GEO
Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.
2016-05-01 | E-MTAB-4143 | biostudies-arrayexpress
Project description:CRISPR-Cas9 has tremendous potential as a therapeutic tool for treating human diseases. However, prolonged expression of the nuclease and gRNA from viral vectors in an in vivo context may cause off-target activity and immunogenicity. While extracellular vesicles have been recently demonstrated to be a promising option to transiently deliver the CRISPR-system, sufficient packaging of both Cas9 protein and gRNA is critical to achieve efficient genome editing in hard-to-transfect cells and tissues, such as skeletal muscle. Here, we developed a novel ribonucleoprotein delivery system utilizing two distinct homing mechanisms. The first is by chemical induced dimerization to recruit Cas9 protein into extracellular nanovesicles. The second utilizes a viral RNA packaging signal and two self-cleaving riboswitches to tether and release sgRNA into nanovesicles. We term our fully engineered delivery system NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo) and demonstrate efficient genome editing in various hard-to-transfect cell types, including human iPS cells and myoblasts. Furthermore, NanoMEDIC production is scalable for industrial production as a xeno-free suspension culture system. As a disease model, therapeutic exon skipping in the dystrophin gene locus was targeted and resulted in over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy patient iPS cells. Finally, we generated novel luciferase-based reporter mice to demonstrate that NanoMEDIC could induce exon skipping and sustain skipping activity for over 160 days, even though NanoMEDIC itself was rapidly degraded within 3 days, indicating its utility for transient in vivo genome editing therapy of DMD and beyond.
2020-01-30 | PXD014527 | JPOST Repository
Project description:Viruses and virally-derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR-Cas9 protein and guide RNA can target genome editing tools to specific cells. These Cas9-packaging enveloped delivery vehicles (Cas9-EDVs), programmed with different displayed antibody fragments, confer genome editing in target cells over bystander cells in mixed cell populations both ex vivo and in vivo. This strategy enabled the generation of genome-edited chimeric antigen receptor (CAR) T cells in humanized mice, establishing a new programmable delivery modality with the potential for widespread therapeutic utility.
2023-06-30 | GSE235643 | GEO
Project description:Delivery strategies for gene editing applications can affect editing outcomes by eliciting distinct cellular responses. This work presents bulk RNA sequencing data from human bronchial epithelial CFF-16HBEge cells, following delivery of an adenine base editor and sgRNA as plasmid DNA via lipofection or as ribonucleoprotein complexes via engineered virus-like particles (eVLPs). Differential gene expression and gene ontology analyses revealed condition-specific transcriptional profiles. This dataset provides a resource for investigating cellular responses to gene editing delivery approaches in vitro.
2026-07-01 | GSE311401 | GEO
Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5’NGG3’ protospacer adjacent motifs (PAM). Examination of dmCas9 binding sites using two Trp53 targeting sgRNAs in Arf -/- MEF cell line (mouse).
2014-08-29 | E-GEOD-57650 | biostudies-arrayexpress
Project description:Virus-mediated expression of CRISPR/Cas9 has been actively used for genome editing in animal models of genetic disorders, including neurological diseases, but the consequences of overexpressing bacterial Cas9 in the mammalian brain remain unknown. Through RNA-seq analysis, we found that virus-mediated expression of Cas9 caused systematic changes in genes involved in neuronal functions. We also generated a short-lived version of Cas9, which maintains its genome editing capacity, but significantly alleviates neurotoxicity caused by overexpressed Cas9. Thus, modification of Cas9 by shortening its half-life would help develop CRISPR/Cas9-based therapeutic approaches for treating genetic neurological disorders.
2018-12-04 | GSE122072 | GEO