Ontology highlight
ABSTRACT:
PROVIDER: PRJNA644281 | ENA |
REPOSITORIES: ENA
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Project description:Gene disruption by CRISPR/Cas9 is highly efficient and relies on the error-prone non-homologous end-joining (NHEJ) pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than NHEJ in mammalian cells. Here, by testing whether manipulation of DNA repair factors would improve HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative 53BP1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem (iPS) cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of NHEJ-mediated DSB repair in the presence of these two factors is not suppressed, and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.
2017-11-23 | GSE103241 | GEO
Project description:Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach to increasing the frequency of HDR in human HSPCs by the delivery of an inhibitor of 53BP1 (named "i53") as a recombinant peptide. We show that the use of i53 peptide effectively increases the frequency of HDR-mediated genome editing at a variety of therapeutically relevant loci in HSPCs as well as other primary human cell types. We show that incorporating the use of i53 recombinant protein allows high frequencies of HDR while lowering the amounts of AAV6 needed by 8-fold. HDR edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in NSG mice, suggesting that i53 recombinant protein might be safely integrated into the standard CRISPR/AAV6-mediated genome editing protocol to gain greater numbers of edited cells for transplantation of clinically meaningful cell populations.
2023-12-31 | GSE246766 | GEO
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:CRISPResso is a software pipeline for the analysis of targeted CRISPR-Cas9 deep sequencing data. This algorithm allows for the quantification of both non-homologous end joining (NHEJ) and homologous directed repair (HDR) occurrences (https://github.com/lucapinello/CRISPResso)
2016-03-04 | GSE78729 | GEO
Project description:Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies suggested that PID phosphorylates and activates PIN1. Here we report unexpected findings about the genetic interactions between the two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9 and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed three independent pid null mutants whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted GFP into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFP HDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFP HDR line also rescued various pid null mutants in a semi-dominant fashion. In addition, the previously reported key phosphorylation sites in PIN1 were still phosphorylated in PIN1-GFP pid plants. We conclude that PID is not directly required for phosphorylation and activation of PIN1.
2023-07-07 | MSV000092373 | MassIVE
Project description:The CRISPR system identified in Streptococcus pyogenes (Sp) has been widely applied in genome editing. In this system, under the direction of gRNA, endonuclease SpCas9 cut both strands of the cognate DNA. These processes may disrupt the open reading frame of the gene and generate a knockout (KO) allele or achieve precise gene knockin (KI). Here we report the dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serum type 6 (AAV6) template in four cell types. We show that editing profiles have distinct differences between cell lines. We reveal AAV6-mediated HDR effectively outcompetes MMEJ-mediated longer deletions but not NHEJ-mediated indels. However, a combination of the small molecule compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to an up to a 3-fold increase in HDR efficiency.
2020-12-14 | GSE155891 | GEO
Project description:APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine to uracil deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR-Cas9. Here, we show in human cells that APOBEC3s can trigger the cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through the homology-directed repair (HDR) of Cas9-generated double-strand breaks . In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks can be further processed to yield mutations mainly involving insertions or deletions (indels). Mechanistically, both APOBEC3-mediated deamination and DNA repair proteins play important roles in the generation of these indels. Correspondingly, optimizing conditions for the repair of CRISPR-Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can substantially repress these unwanted mutations in genomic DNA.
2017-11-10 | GSE105146 | GEO