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HDR-CRISPR promotes targeted genome editing with reduced mutational burden in primary human cells [CAST-seq]

ABSTRACT: We have established a technology to endorse DSB resolution via HDR with the goal of facilitating clinical transition of HDR-based editing strategies and increase safety. We have used high throughput sequencing as deposited here to assess safety of our platform

ORGANISM(S): Homo sapiens

PROVIDER: GSE225449 | GEO | 2023/08/11

REPOSITORIES: GEO

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HDR-CRISPR promotes targeted genome editing with reduced mutational burden in primary human cells [Amp-seq]

Project description:We have established a technology to endorse DSB resolution via HDR with the goal of facilitating clinical transition of HDR-based editing strategies and increase safety. We have used high throughput sequencing as deposited here to assess safety of our platform

2023-08-11 | GSE225450 | GEO

HDR-CRISPR promotes targeted genome editing with reduced mutational burden in primary human cells

Project description:This SuperSeries is composed of the SubSeries listed below.

2023-08-11 | GSE225452 | GEO

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Project description:Panicum virgatum HDR Resequencing genome sequencing

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RAD51AP1 regulates ALT-HDR through chromatin-directed homeostasis of TERRA

Project description:Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of highly aggressive cancer. Recent studies have revealed that TERRA (telomere repeat-containing RNA) acts to initiate ALT-associated HDR (ALT-HDR). Here we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R- loop HR intermediates. We also show that RAD51AP1 binds to and may generate and potentially stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a new role for RAD51AP1 mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide new insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.

2024-01-26 | PXD035943 | Pride

Characterization of tumor mutational burden in LKB1 mutant lung tumors

Project description:Inactivating mutations in LKB1/STK11 are present in ~20% of non-small cell lung cancers (NSCLC) and portend poor response to anti-PD-1 immunotherapy in patients and genetically engineered mouse model (GEMMs). Here, we sought to uncover the basis for immunotherapy resistance of these tumors and to define strategies that overcome this barrier. Whereas high tumor mutational burden (TMB) often correlates with response to anti-PD1 treatment, we found that LKB1-deficient NSCLCs from non-smokers and GEMMs exhibited striking elevations in nonsynonymous mutations compared to LKB1 wildtype tumors. Correspondingly, LKB1 mutant NSCLC cell lines showed defects in both replication dependent and independent double-strand DNA break (DSB) repair, which were reversed upon LKB1 re-expression.

2021-03-17 | GSE137244 | GEO

Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR/Cas9 genome editing

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

DMC1 ChIP-seq from B6xCAST F1 PRDM9-Humanized/CAST testes

Project description:We report testis DMC1 enrichment at DSB (Double-Strand Break) hotspots in a B6xCAST F1 mouse, which is heterozygous at Prdm9, with one wild-type CAST allele and one allele found in human populations (bred from genetically engineered mother, Davies et al Nature 2012). We also report H3K4me3 intensities at these hotspots, based on the raw data published under GSE119727 (Li et al). Separately, we have reported 2,649 crossover locations identified by single-cell DNA sequencing of 217 sperm from the same hybrid mouse. We identify several factors, including PRDM9 binding on the repair-template homologue, telomere proximity and local GC-content, that affect the probability that a DSB is repaired as a crossover. We further show that these factors also influence the time it takes for the site of a DSB to find and engage its homologue, with rapidly-engaging sites being more likely to be repaired as crossovers.

2019-03-18 | GSE124991 | GEO

RNA-Seq for validating the RNA expression of wild type and analog sensitive CDK12 and CDK13 in analog sensitive MV4;11 cell lines that generated by CRISPR HDR

Project description:We used Poly-A enriched RNA-Seq to validate CDK12, CDK13 anlalog senstive mutationsand their expression level in the cell lines that has analog sensitive mutations that were introduced by CRISPR HDR

2020-04-29 | GSE141366 | GEO

Crossovers from B6xCAST F1 PRDM9-Humanized/CAST testes

Project description:We report 2,649 crossover breakpoints identified by single-cell DNA sequencing of 217 sperm in a B6xCAST F1 mouse, which is heterozygous at Prdm9, with one wild-type CAST allele and one allele found in human populations (bred from a genetically engineered mother, Davies et al Nature 2012). Separately, we have reported testis DMC1 enrichment in the same mouse (GSE124991). We also infer H3K4me3 intensities at DMC1 hotspots, based on the raw H3K4me3 ChIP-seq data published under GSE119727 (Li et al). We identify several factors, including PRDM9 binding on the repair-template homologue, telomere proximity and local GC-content, that affect the probability that a DSB is repaired as a crossover. We further show that these factors also influence the time it takes for the site of a DSB to find and engage its homologue, with rapidly-engaging sites being more likely to be repaired as crossovers.

2019-03-18 | GSE125326 | GEO