scholarly journals Generation of scalable cancer models by combining AAV-intron-trap, CRISPR/Cas9, and inducible Cre-recombinase

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Prajwal C. Boddu ◽  
Abhishek K. Gupta ◽  
Jung-Sik Kim ◽  
Karla M. Neugebauer ◽  
Todd Waldman ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mutant Allele ◽  
Cre Recombinase ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Dna Breaks ◽  
Genomic Locus ◽  
Trap Design ◽  
Double Stranded Dna ◽  
Cancer Models

AbstractScalable isogenic models of cancer-associated mutations are critical to studying dysregulated gene function. Nonsynonymous mutations of splicing factors, which typically affect one allele, are common in many cancers, but paradoxically confer growth disadvantage to cell lines, making their generation and expansion challenging. Here, we combine AAV-intron trap, CRISPR/Cas9, and inducible Cre-recombinase systems to achieve >90% efficiency to introduce the oncogenic K700E mutation in SF3B1, a splicing factor commonly mutated in multiple cancers. The intron-trap design of AAV vector limits editing to one allele. CRISPR/Cas9-induced double stranded DNA breaks direct homologous recombination to the desired genomic locus. Inducible Cre-recombinase allows for the expansion of cells prior to loxp excision and expression of the mutant allele.  Importantly, AAV or CRISPR/Cas9 alone results in much lower editing efficiency and the edited cells do not expand due to toxicity of SF3B1-K700E. Our approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes confer a growth disadvantage.

scholarly journals DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Prasun Chakraborty ◽  
Kevin Hiom
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Recombination Repair ◽  
Dna End Resection ◽  
End Resection ◽  
Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

scholarly journals Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break

2019 ◽  
Author(s):  
Geoffrey L. Rogers ◽  
Hsu-Yu Chen ◽  
Heidy Morales ◽  
Paula M. Cannon
Keyword(s):  
Homologous Recombination ◽  
Progenitor Cells ◽  
Genome Editing ◽  
High Efficiency ◽  
Zinc Finger Nucleases ◽  
Dna Breaks ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem ◽  
Double Stranded Dna ◽  
Dna Break

AbstractAdeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem/progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 or AAVS1 loci in human HSPCs and cell lines. As a control we used AAV6, which effectively edits HSPCs, but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results therefore do not support that clade F AAVs can perform high efficiency genome editing in the absence of a DSB.

scholarly journals RecA finds homologous DNA by reduced dimensionality search

Nature ◽  
2021 ◽  
Author(s):  
Jakub Wiktor ◽  
Arvid H. Gynnå ◽  
Prune Leroy ◽  
Jimmy Larsson ◽  
Giovanna Coceano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Fluorescence Microscopy ◽  
Single Cells ◽  
Search Time ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Sister Chromosome ◽  
Repair Template ◽  
Living Organisms

AbstractHomologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3′ single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.

scholarly journals Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (7) ◽  
pp. 4493-4500
Author(s):  
R H Schiestl ◽  
J Zhu ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Restriction Enzyme ◽  
Strand Break ◽  
Illegitimate Recombination ◽  
Dna Breaks ◽  
Homologous Integration ◽  
Double Stranded Dna ◽  
Fold Reduction

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these recombination events. Surprisingly, REM events are completely independent of the double-strand-break repair functions encoded by the RAD51, RAD52, and RAD57 genes but require the RAD50 gene product. IR events are under different genetic control than homologous integration events. In the rad50 mutant, homologous integration occurred at wild-type frequency, whereas the frequency of IR events was 20- to 100-fold reduced. Conversely, the rad52 mutant was grossly deficient in homologous integration (at least 1,000-fold reduced) but showed only a 2- to 8-fold reduction in IR frequency.

scholarly journals 5-Fluorouracil sensitizes colorectal tumor cells towards double stranded DNA breaks by interfering with homologous recombination repair

Oncotarget ◽  
2015 ◽  
Vol 6 (14) ◽  
pp. 12574-12586 ◽  
Author(s):  
Upadhyayula Sai Srinivas ◽  
Jerzy Dyczkowski ◽  
Tim Beißbarth ◽  
Jochen Gaedcke ◽  
Wael Y. Mansour ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Tumor Cells ◽  
Colorectal Tumor ◽  
Homologous Recombination Repair ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Recombination Repair

RecA family proteins in archaea: RadA and its cousins

2009 ◽  
Vol 37 (1) ◽  
pp. 102-107 ◽  
Author(s):  
Sam Haldenby ◽  
Malcolm F. White ◽  
Thorsten Allers
Keyword(s):  
Phylogenetic Analysis ◽  
Dna Replication ◽  
Homologous Recombination ◽  
Genome Stability ◽  
Model Organisms ◽  
Replication Forks ◽  
Dna Breaks ◽  
Double Stranded Dna

Recombinases of the RecA family are essential for homologous recombination and underpin genome stability, by promoting the repair of double-stranded DNA breaks and the rescue of collapsed DNA replication forks. Until now, our understanding of homologous recombination has relied on studies of bacterial and eukaryotic model organisms. Archaea provide new opportunities to study how recombination operates in a lineage distinct from bacteria and eukaryotes. In the present paper, we focus on RadA, the archaeal RecA family recombinase, and its homologues in archaea and other domains. On the basis of phylogenetic analysis, we propose that a family of archaeal proteins with a single RecA domain, which are currently annotated as KaiC, be renamed aRadC.

scholarly journals Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (7) ◽  
pp. 4493-4500 ◽  
Author(s):  
R H Schiestl ◽  
J Zhu ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Restriction Enzyme ◽  
Strand Break ◽  
Illegitimate Recombination ◽  
Dna Breaks ◽  
Homologous Integration ◽  
Double Stranded Dna ◽  
Fold Reduction

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these recombination events. Surprisingly, REM events are completely independent of the double-strand-break repair functions encoded by the RAD51, RAD52, and RAD57 genes but require the RAD50 gene product. IR events are under different genetic control than homologous integration events. In the rad50 mutant, homologous integration occurred at wild-type frequency, whereas the frequency of IR events was 20- to 100-fold reduced. Conversely, the rad52 mutant was grossly deficient in homologous integration (at least 1,000-fold reduced) but showed only a 2- to 8-fold reduction in IR frequency.

Suppressors of a Cold-Sensitive Mutation in Yeast U4 RNA Define Five Domains in the Splicing Factor Prp8 That Influence Spliceosome Activation

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1667-1682 ◽  
Author(s):  
Andreas N Kuhn ◽  
David A Brow
Keyword(s):  
Large Scale ◽  
Splicing Factor ◽  
Cold Sensitivity ◽  
Splicing Factors ◽  
Loss Of Function ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Cold Sensitive ◽  
Spliceosome Activation ◽  
U4 Rna

AbstractThe highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

scholarly journals Germline Mutations in Other Homologous Recombination Repair-Related Genes Than BRCA1/2: Predictive or Prognostic Factors?

2021 ◽  
Vol 11 (4) ◽  
pp. 245
Author(s):  
Laura Cortesi ◽  
Claudia Piombino ◽  
Angela Toss
Keyword(s):  
Prognostic Factors ◽  
Homologous Recombination ◽  
Pancreatic Adenocarcinoma ◽  
Objective Response ◽  
Metastatic Breast ◽  
Germline Mutations ◽  
Response To Treatment ◽  
Homologous Recombination Repair ◽  
Dna Breaks ◽  
Recombination Repair

The homologous recombination repair (HRR) pathway repairs double-strand DNA breaks, mostly by BRCA1 and BRCA2, although other proteins such as ATM, CHEK2, and PALB2 are also involved. BRCA1/2 germline mutations are targeted by PARP inhibitors. The aim of this commentary is to explore whether germline mutations in HRR-related genes other than BRCA1/2 have to be considered as prognostic factors or predictive to therapies by discussing the results of two articles published in December 2020. The TBCRC 048 trial published by Tung et al. showed an impressive objective response rate to olaparib in metastatic breast cancer patients with germline PALB2 mutation compared to germline ATM and CHEK2 mutation carriers. Additionally, Yadav et al. observed a significantly longer overall survival in pancreatic adenocarcinoma patients with germline HRR mutations compared to non-carriers. In our opinion, assuming that PALB2 is a high-penetrant gene with a key role in the HRR system, PALB2 mutations are predictive factors for response to treatment. Moreover, germline mutations in the ATM gene provide a better outcome in pancreatic adenocarcinoma, being more often associated to wild-type KRAS. In conclusion, sequencing of HRR-related genes other than BRCA1/2 should be routinely offered as part of a biological characterization of pancreatic and breast cancers.

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