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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A multi-functional AAV-CRISPR-Cas9 and its host response

ABSTRACT: 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).

ORGANISM(S): Mus musculus

SUBMITTER: Alex Ng

PROVIDER: E-GEOD-84534 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A multi-functional AAV-CRISPR-Cas9 and its host response

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

2016-07-29 | GSE84534 | GEO

KDM5C safeguards the appropriate timing of early neurodevelopment

Project description:We generated human induced pluripotent cells from intellectual disability patients carrying the c.2T>C mutation in KDM5C (Called “Mutant”). We generated a paired, isogenic human iPS cell line (called “Corrected”) using CRISPR/Cas9 and PiggyBac gene-editing technologies and conducted neuronal differentiation based on “Yichen Shi et al. Nat. Protoc. 7, 1836–1846 (2012)” to define differences in gene expression between the Mutant and Corrected during neurodevelopment.

2023-10-13 | E-MTAB-7551 | biostudies-arrayexpress

Safeguarding genome integrity during gene-editing therapy of age-related macular degeneration

Project description:Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model. AAV delivery of both CRISPR-Cas9 and CRISPR-Cas9TX can efficiently inhibit laser-induced neovascularization. Importantly, Cas9TX almost eliminates chromosomal translocations that occur at a frequency of approximately 1% in Cas9-edited mouse retinal cells. Strikingly, the widely observed AAV integration at the target Vegfa site is also greatly dropped from nearly 50% of edited events to the background level during Cas9TX editing. Our findings reveal that chromosomal structural variations routinely occur during in vivo genome editing and highlight Cas9TX as a superior form of Cas9 for in vivo gene disruption.

2022-11-30 | GSE218818 | GEO

rRNA 2'-O-methylation profile of SNORD71 knockdown cells

Project description:We generated a SNORD71 KO chondrocyte cell pool using CRISPR/Cas9 gene editing. A CRISPR control cell line was generated and used as a control. Levels of 2’-O-methylation of human rRNAs in SNORD71 KO cell pool and CRISPR control cells were evaluated by RiboMethSeq.

2022-03-05 | E-MTAB-11470 | biostudies-arrayexpress

RNA-guided Location and Function of the Cas9 CRISPR Endonuclease in Mammalian Cells

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

Transcriptome changes triggered by the loss of function of gene Mppcs in genome-edited Marchantia polymorpha plants

Project description:In the present study, Marchantia polymorpha Mppcs loss of function mutants were generated through CRISPR/cas9 mediated genome-editing. To assess whether the knockout of MpPCS gene affects the transcription of M. polymorpha nuclear genes in unstressed condition, the Mppcs-2 knockout mutant and Cam2 wild-type transcriptomes were compared by RNA-Seq.

2022-08-29 | E-MTAB-11885 | biostudies-arrayexpress

miRNA-seq of HEK293 cell lines on Illumina iSeq100 instrument

Project description:A validation experiment performed on HEK293 cell lines after genome editing. The design contains three duplicate runs consisted of: HEK293 wild type cell line HEK293 with MIR484 gene knockdown using CRISPR-Cas9 HEK293 with MIR185 gene knockout using CRISPR-Cas9

2023-06-16 | E-MTAB-13009 | biostudies-arrayexpress

Proteome analysis of NanoMEDIC particles

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

Multi-species RELA ChIP-seq of mammalian aortic endothelial cells before and after TNFα stimulation

Project description:TNFα has an evolutionary conserved role in mediating inflammation via activation of the transcription factor NF-κB. The functions of individual NF-κB binding sites are not well understood. To identify conserved and functionally important NF-κB binding sites in mammals, we performed ChIP-seq to map the genome-wide binding of RELA and select histone modifications in primary vascular endothelial cells (ECs) isolated from the aortas of human (HAEC), mouse (MAEC) and cow (BAEC), before and after TNFα. The conserved RELA binding sites show strong epigenetic changes in response to TNFα and enrich near genes controlling vascular development and pro-inflammatory responses. Our method identifies novel modes of RELA-chromatin interactions that are conserved in mammals and shared between multiple cell-types. Particularly, genomic regions bound by RELA prior to stimulation are important responders during TNFα stimulation. We use CRISPR/Cas9 genome editing to validate the roles of the conserved RELA pre-bound sites near pro-inflammatory genes such as CCL2 and PLK2. Our evolutionary approach describes new aspects of mammalian NF-κB biology including its role within super-enhancers and relevance in inflammatory disorders.

2020-10-03 | E-MTAB-7889 | biostudies-arrayexpress

Treatment of a metabolic liver disease by in vivo prime editing in mice

Project description:Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of genetic defects1. However, correction of a disease phenotype in vivo in somatic tissues has not been achieved yet. Here, we establish proof-of-concept for in vivo prime editing, that resulted in rescue of a metabolic liver disease. We first develop a size-reduced prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (SpCas9-PERnH), and a linker- and NLS-optimized intein-split PE construct (SpCas9-PE p.1153) for delivery by adeno-associated viruses (AAV). Systemic dual AAV-mediated delivery of this variant in neonatal mice enables installation of a transversion mutation at the Dnmt1 locus with 15% efficiency on average. Next, we targeted the disease-causing mutation in the phenylalanine hydroxylase (Pah)enu2 mouse model for phenylketonuria (PKU). Correction rates of 1.5% using the dual AAV approach could be increased to up to 14% by delivery of full-length SpCas9-PE via adenoviral vector 5 (AdV5), leading to full restoration of physiological blood phenylalanine (L-Phe) levels below 120 µmol/L. Our study demonstrates in vivo prime editing in the liver at two independent loci, emphasizing the potential of PEs for future therapeutic applications.

2021-12-29 | GSE174757 | GEO

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