Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA850001 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Haplotype-phased Callithrix jacchus embryonic stem cell line for genome editing using CRISPR/Cas9
2018-07-09 | PRJEB27676 | EVA
Project description:CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2,500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.
2022-06-07 | GSE178440 | GEO
Project description:Chronic granulomatous disease (CGD) is a severe inborn error of immunity (IEI) caused by defects in the NADPH oxidase. The best current treatment option for patients with CGD, allogeneic hematopoietic stem cell (HSC) transplantation, is associated with severe adverse effects such as graft-versus-host disease, highlighting a need for improved treatment options based on transplantation of autologous ex vivo gene-edited HSCs. Here, we generate CRISPR/Cas9-based gene editing strategies for the correction of two CGD-causing variants; CYBA c.287+1G>T (p22phox) causing autosomal CGD and CYBB c.252G>A (NOX2) causing X-linked CGD. We find that rAAV6 outperforms ssODNs and IDLVs as HDR repair templates in CD34+ hematopoietic stem and progenitor cells (HSPCs), and we optimize gene editing strategies further by including mRNA-encoded inhibitors/effectors. In addition, we develop a near-universal gene editing strategy for X-CGD by targeted integration of a truncated CYBB cDNA, covering 86% of X-CGD patients, and show functional ROS production in edited cells. We find prevalent off-target editing and chromosomal translocations that severely decreased the ability of gene edited HSPCs to engraft in immunodeficient mice, however, by limiting off-target editing through the use of a high-fidelity Cas9, we can show that we can substantially rescue the multilineage engraftment potential of the gene edited HSPCs. To further improve safety, we finally we develop a novel paired D10A Cas9n gene editing approach targeting CYBB. We demonstrate that using this D10A Cas9 approach, we can retain high on-target efficacy while we do not detect any off-target editing or translocations between on- or off-target cleavage sites. Collectively, we bring new insights into to safety and off-target effects of gene editing approaches targeting CYBA and CYBB, highlighting key challenges of these approaches while offering a potentially curative D10A Cas9n-based treatment option for patients with CGD with improved safety.
2025-02-24 | GSE287370 | GEO
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:High-fidelity Cas enzyme is pivotal to the safety of CRISPR technology in translational research and clinics. However, increasing editing fidelity often comes at the cost of a significant decrease in nuclease editing efficiency, e.g., the rationale designed SuperFi-Cas9 based on the structural basis for mismatch surveillance of wildtype Cas9. By conducting high-throughput profiling on the editing efficiency of 91,603 gRNAs, we found that SuperFi-Cas9/gRNA showed a strong nucleotide preference at the 21st position of the PAM-distal region of the target. A deep-learning model trained from the profiling data further provided a mutation map, which implied that a gRNA-target duplex with twenty-one matched nucleotides positively contributed to the editing efficiency of SuperFi-Cas9/gRNA. By further measuring the cleavage activities at twenty endogenous genomic locations, we demonstrated that a gRNA with extended 5' nucleotides significantly increased the editing activity of SuperFi-Cas9 while remaining its high fidelity, which makes SuperFi-Cas9 a valuable nuclease of the CRISPR toolbox. Together, by deep-learning modeling on high throughput profiling data, we reported that gRNAs with extended 5' nucleotides can rescue the impaired cleavage activities of SuperFi-Cas9.
2025-12-15 | GSE279806 | GEO
Project description:The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypic Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated for the first time the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.
2024-02-09 | GSE249731 | GEO
Project description:Recurring deletions of chromosome 7 and 7q [-7/del(7q)] occur in myelodysplastic syndromes and acute myeloid leukemia (AML) and are associated with poor prognosis. However, the identity of specific tumor suppressors on 7q remains elusive. Using RNAi and CRISPR/Cas9 approaches, we show that a ~50% reduction in gene dosage of the mixed lineage leukemia 3 (MLL3) gene, located on 7q36.1, cooperates with other events occurring in -7/del(7q) AMLs to promote leukemogenesis. Mll3 suppression impairs the differentiation of HSPC. Interestingly, Mll3 suppressed leukemias, like human -7/del(7q) AMLs, are refractory to conventional chemotherapy but sensitive to the BET inhibitor JQ1. Thus, our mouse model functionally validates MLL3 as a haploinsufficient 7q tumor suppressor, and suggests a therapeutic option for this aggressive disease. Total RNA obtained from sorted lin-ckit+ hematopoietic stem and progenitor cells of recipient mice transplanted with shRen;p53-/- or shMll3;p53-/- cells at 6 weeks after transplant
2014-03-19 | E-GEOD-54313 | biostudies-arrayexpress
Project description:We have developed a therapeutic strategy for beta-hemoglobinopathies aimed at reactivating fetal hemoglobin expression in red blood cells derived from human hematopoietic stem/progenitor cells edited with CRISPR/Cas9 nucleases, cytidine or adenine base editors targeting the fetal gamma-globin promoters. Here, we report the transcriptomic changes occurring in human hematopoietic stem/progenitor cells (obtained from healthy donors) 48 h after transfection with CRISPR/Cas9 nucleases, cytidine or adenine base editors.
2022-09-06 | GSE191135 | GEO
Project description:New method to measure tumor growth with programmable multiplexed CRISPR/Cas9-induced genotypes via ultra-deep sequencing of DNA barcoded tumors
2017-05-10 | GSE98207 | GEO