Ontology highlight
ABSTRACT:
PROVIDER: PRJNA665073 | ENA |
REPOSITORIES: ENA
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Project description:Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.
2024-07-30 | GSE272812 | GEO
Project description:This study aims to predict the activity and specificity of CRISPR/Cas9 by deep learning at genome-scale among different cell lines. Here, we have focused on embracing and modifying a system for evaluating SpCas9 activity of on-target and off-target using >1,000,000 guide RNAs (gRNAs) covering ~20,000 protein-coding genes and ~10,000 non-coding genes in synthetic constructs with a high-throughput manner. With the help of deep learning algorithms in the field of artificial intelligence, three prediction models with the best generalization performance now are constructed: Aidit_Cas9-ON, Aidit_Cas9-OFF, and Aidit_Cas9-DSB. Moreover, through systematically investigating the influence of diverse cellular environment on gRNA activity and specificity, we noticed that distinct features are favored from H1 cell line compared with the other 2 cell lines for on-target activity and the overall distribution of repair outcomes is markedly different across 3 cell lines, especially in Jurkat. Finally, we identify a key effect protein DNTT strongly influences editing outcomes induced by CRISPR/Cas9. We confirm that this study will greatly facilitate CRISPR-based genome editing.
2023-05-24 | GSE181774 | GEO
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: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
Project description:CRISPR-Cas constitutes an adaptive prokaryotic defence system against invasive nucleic acids like viruses and plasmids. Beyond their role in immunity, CRISPR-Cas systems have been shown to closely interact with components of cellular DNA repair pathways either by regulating their expression or via direct protein-protein contact or enzymatic activity. The integrase Cas1 is usually involved in the adaptation phase of CRISPR-Cas immunity but its function in cellular DNA repair pathways has been proposed before. Here, we analysed the capacity of an archaeal Cas1 from Haloferax volcanii to compensate DNA damage induced by oxidative stress and found that a deletion of the cas1 gene led to severe growth defects after stress induction. In addition, our results indicate that Cas1 is directly involved in DNA damage repair as the enzymatically active site of the protein is crucial for growth rescue under oxidative conditions. Based on biochemical cleavage assays, we propose a mechanism in which Cas1 exerts a similar function like the DNA repair protein Fen1 by resolving branched repair intermediate structures. Overall, the present study broadens our understanding of the functional link between CRISPR-Cas immunity and DNA repair by demonstrating that Cas1 and Fen1 display commutable roles during archaeal DNA damage repair.
2022-05-20 | PXD029952 | Pride
Project description:Cornelia de Lange syndrome (CdLS) is an autosomal dominant disease mainly caused by mutations in the Nipped-B-like protein (NIPBL) gene resulting in the alteration of the cohesin pathway. Here, we generated human induced pluripotent stem cells (hiPSCs) from a CdLS patient carrying a mutation in the NIPBL gene, c.5483G>A, and tested CRISPR-Cas based approaches to repair the genetic defect. We applied an efficient and precise method of gene correction through CRISPR-Cas induced homology directed repair (HDR), which allowed the generation of hiPSC clones with regular karyotype and preserved stemness. The efficient and precise gene replacement strategy developed in this study can be extended to the modification of other genomic loci in hiPSCs. Isogenic wild-type and mutated hiPSCs produced with the CRISPR-Cas technology are fundamental CdLS cellular models to study the disease molecular determinants and identifying therapeutic targets.
2023-03-17 | E-MTAB-11604 | biostudies-arrayexpress
Project description:A Scalable Epitope Tagging Approach for High Throughput ChIP-seq Analysis ChIP-seq comparison between CRISPR editing cells using epitope antibody and non-editing cells using endogeneous TF antibody
2016-09-01 | E-GEOD-78064 | biostudies-arrayexpress
Project description:The gain-of-function mutation of c.G6055A (p.G2019S) in Leucine-rich repeat kinase 2 (LRRK2) gene is the most prevalent genetic cause of Parkinson’s disease (PD). Although clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has been used to generate isogenic control lines, there are risks of creating indels and genomic instability together with a lower efficiency in non-dividing cells. The recent advance of adenine base editors (ABEs) could convert targeted A•T base pairs to G•C base pairs without double-strand DNA breaks or donor DNA templates and function in post-mitotic cells. Here, we demonstrate a complete correction of an induced pluripotent stem cell (iPSC) line derived from a PD patient having LRRK2 p.G2019S mutation using variable genome editing methods, including CRISPR/Cas9-based homology-directed repair (HDR) and ABEs. The corrected isogenic iPSCs derived dopaminergic neurons showed a down-regulated LRRK2 kinase activity, decreased phospho-a-synuclein accumulations, reduced apoptosis and restored neurite shrinkage phenotypes. The mutation correction efficacy, off-target and indels rates between CRISPR/Cas9-HDR and ABE were compared. Among the 47 clones generated by HDR, 3 clones (6.4%) were on-targeted corrected, while ABE showed a much higher correction rate (13 of 53 clones, 24.5%). Whole genome sequencing analysis revealed that there are 27 clones of HDR (57.4%) having deletions but none in clones of ABE, albeit ABE created 14 clones (26.4%) having off-target missense mutations. RNA sequencing and proteomic analysis of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of PD relevant pathways, including calcium ion dependent exocytosis, synaptic transports well as potential novel targets relevant to PD pathophysiology. These results envision that ABE could directly correct the pathogenic PD mutation in iPSCs for exploring the earliest events in PD pathophysiology and providing dopaminergic neurons for future cell therapies.
2022-04-24 | PXD025602 | JPOST Repository