Transient Inhibition of 53BP1 Increases the Frequency of Targeted Integration in Human Hematopoietic Stem and Progenitor Cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1034575 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26628883_1.fastq.gz | Fastqsanger.gz | |||
| SRR26628883_2.fastq.gz | Fastqsanger.gz | |||
| SRR26628884_1.fastq.gz | Fastqsanger.gz | |||
| SRR26628884_2.fastq.gz | Fastqsanger.gz | |||
| SRR26628885_1.fastq.gz | Fastqsanger.gz |
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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: Not available
| S-EPMC10762240 | biostudies-literature
Project description:RAG endonuclease initiates V(D)J recombination by cleaving recombination signal sequences (RSSs), subsequently repaired by classical nonhomologous end-joining (c-NHEJ). However, 53BP1, as a key DNA damage response (DDR) factor promoting c-NHEJ, is dispensable for V(D)J recombination. Here, we report that 53BP1 orchestrates sequence strength of RAG targets to shape end-joining features during V(D)J recombination. Deficiency of 53BP1, rather than other DDR factors, specifically increases junctional microhomology (MH) of cryptic RSS (cRSS) recombination instead of RSS recombination. Depletion of RIF1 or Shieldin, downstream factors of 53BP1, partially mimics the phenotypes of 53BP1-deficiency. Notably, loss of RNF168, rather than other classical resection and alternative end-joining factors, is sufficient to fully rescue 53BP1-deficiency-mediated increase of junctional MH during cRSS recombination. Remarkably, RAG mutant and RSS score analyses indicate that sequence strength of RAG targets determines the unique roles of 53BP1 in balancing end-joining outcomes. Our findings provide deeper mechanistic insights into DNA damage repair for RAG cleaved on- and off-targets during V(D)J recombination.
2026-04-09 | GSE270090 | GEO
Project description:RAG endonuclease initiates V(D)J recombination by cleaving recombination signal sequences (RSSs), subsequently repaired by classical nonhomologous end-joining (c-NHEJ). However, 53BP1, as a key DNA damage response (DDR) factor promoting c-NHEJ, is dispensable for V(D)J recombination. Here, we report that 53BP1 orchestrates sequence strength of RAG targets to shape end-joining features during V(D)J recombination. Deficiency of 53BP1, rather than other DDR factors, specifically increases junctional microhomology (MH) of cryptic RSS (cRSS) recombination instead of RSS recombination. Depletion of RIF1 or Shieldin, downstream factors of 53BP1, partially mimics the phenotypes of 53BP1-deficiency. Notably, loss of RNF168, rather than other classical resection and alternative end-joining factors, is sufficient to fully rescue 53BP1-deficiency-mediated increase of junctional MH during cRSS recombination. Remarkably, RAG mutant and RSS score analyses indicate that sequence strength of RAG targets determines the unique roles of 53BP1 in balancing end-joining outcomes. Our findings provide deeper mechanistic insights into DNA damage repair for RAG cleaved on- and off-targets during V(D)J recombination.
2026-04-09 | GSE270088 | GEO
Project description:53BP1 activity drives genome instability and embryonic lethality in BRCA1-deficient cells by inhibiting homologous recombination (HR).53BP1’s anti-recombinogenic functions require phosphorylation-dependent interactions with two effector complexes, PTIP and RIF1/Shieldin. While RIF1/Shieldin is thought to block 5’-3’ nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here we show that mutation of the PTIP interaction site in 53BP1 (S25A) increases Shieldin association with DNA damage. Despite excessive Shieldin “end-blocking” activity, the mutant protein allows end resection sufficient to rescue the lethality of BRCA1D11 mice. End resection in BRCA1D1153BP1S25A mice is rewired in a manner driven by DNA2 since Shieldin blocks EXO1-mediated nucleolytic processing. Despite ample resection, mutant cells fail to complete HR, as 53BP1/Shieldin also inhibits RNF168-mediated loading of PALB2/RAD51 onto ssDNA post-resection. As a result, BRCA1D1153BP1S25A mice exhibit hallmark features of HR insufficiency, including increased developmental neuronal apoptosis, premature aging and hypersensitivity to PARP inhibitors. Disruption of Shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study supports a model in which RIF1/Shieldin and PTIP associate independently with 53BP1 to regulate distinct end-resection pathways, and reveals a critical function of 53BP1/Shieldin post-resection that limits RNF168-mediated loading of RAD51.
2019-10-22 | GSE133806 | GEO
Project description:53BP1 activity drives genome instability and embryonic lethality in BRCA1-deficient cells by inhibiting homologous recombination (HR).53BP1’s anti-recombinogenic functions require phosphorylation-dependent interactions with two effector complexes, PTIP and RIF1/Shieldin. While RIF1/Shieldin is thought to block 5’-3’ nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here we show that mutation of the PTIP interaction site in 53BP1 (S25A) increases Shieldin association with DNA damage. Despite excessive Shieldin “end-blocking” activity, the mutant protein allows end resection sufficient to rescue the lethality of BRCA1D11 mice. End resection in BRCA1D1153BP1S25A mice is rewired in a manner driven by DNA2 since Shieldin blocks EXO1-mediated nucleolytic processing. Despite ample resection, mutant cells fail to complete HR, as 53BP1/Shieldin also inhibits RNF168-mediated loading of PALB2/RAD51 onto ssDNA post-resection. As a result, BRCA1D1153BP1S25A mice exhibit hallmark features of HR insufficiency, including increased developmental neuronal apoptosis, premature aging and hypersensitivity to PARP inhibitors. Disruption of Shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study supports a model in which RIF1/Shieldin and PTIP associate independently with 53BP1 to regulate distinct end-resection pathways, and reveals a critical function of 53BP1/Shieldin post-resection that limits RNF168-mediated loading of RAD51.
2019-10-22 | GSE133805 | GEO
Project description:RAG endonuclease initiates V(D)J recombination by cleaving recombination signal sequences (RSSs), subsequently repaired by classical nonhomologous end-joining (c-NHEJ). However, 53BP1, as a key DNA damage response (DDR) factor promoting c-NHEJ, is dispensable for V(D)J recombination. Here, we report that 53BP1 orchestrates sequence strength of RAG targets to shape end-joining features during V(D)J recombination. Deficiency of 53BP1, rather than other DDR factors, specifically increases junctional microhomology (MH) of cryptic RSS (cRSS) recombination instead of RSS recombination. Depletion of RIF1 or Shieldin, downstream factors of 53BP1, partially mimics the phenotypes of 53BP1-deficiency. Notably, loss of RNF168, rather than other classical resection and alternative end-joining factors, is sufficient to fully rescue 53BP1-deficiency-mediated increase of junctional MH during cRSS recombination. Remarkably, RAG mutant and RSS score analyses indicate that sequence strength of RAG targets determines the unique roles of 53BP1 in balancing end-joining outcomes. Our findings provide deeper mechanistic insights into DNA damage repair for RAG cleaved on- and off-targets during V(D)J recombination.
2026-04-09 | GSE270086 | GEO
Project description:RAG endonuclease initiates V(D)J recombination by cleaving recombination signal sequences (RSSs), subsequently repaired by classical nonhomologous end-joining (c-NHEJ). However, 53BP1, as a key DNA damage response (DDR) factor promoting c-NHEJ, is dispensable for V(D)J recombination. Here, we report that 53BP1 orchestrates sequence strength of RAG targets to shape end-joining features during V(D)J recombination. Deficiency of 53BP1, rather than other DDR factors, specifically increases junctional microhomology (MH) of cryptic RSS (cRSS) recombination instead of RSS recombination. Depletion of RIF1 or Shieldin, downstream factors of 53BP1, partially mimics the phenotypes of 53BP1-deficiency. Notably, loss of RNF168, rather than other classical resection and alternative end-joining factors, is sufficient to fully rescue 53BP1-deficiency-mediated increase of junctional MH during cRSS recombination. Remarkably, RAG mutant and RSS score analyses indicate that sequence strength of RAG targets determines the unique roles of 53BP1 in balancing end-joining outcomes. Our findings provide deeper mechanistic insights into DNA damage repair for RAG cleaved on- and off-targets during V(D)J recombination.
2026-04-09 | GSE270092 | GEO
Project description:Absence of 53BP1 influences the profile of DNA rearrangements in B lymphocytes.To determine whether the differences in translocation partner choice were due to differences in transcription, we compared the transcriptome of 53BP1 deficient and wild-type B cells by RNA-Seq and Polymerase II chromatin immunoprecipitation sequencing (ChIP-Seq). Analysis of total mRNA and PolII loading in the presence or absence of 53BP1 in activated B lymphocytes
2013-02-12 | E-GEOD-42350 | biostudies-arrayexpress
Project description:53BP1 is a well-established DNA damage repair factor that has recently been shown to regulate gene expression and critically influence tumor suppression and neural development. For gene regulation, how 53BP1 is regulated remains unclear. Here, we showed that 53BP1-serine 25 phosphorylation by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical organoids. 53BP1-serine 25 phosphorylation dynamics controls 53BP1 target genes for neuronal differentiation and function, cellular response to stress, and apoptosis. Beyond 53BP1, ATM is required for phosphorylation of factors in neuronal differentiation, cytoskeleton, p53 regulation, and ATM, BNDF, and WNT signaling pathways for cortical organoid differentiation. Overall, our data suggest that 53BP1 and ATM control key genetic programs required for human cortical development.
2024-08-11 | GSE231321 | GEO