Elevated Homologous Recombination Activity in Fanconi Anemia Fibroblasts

The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore the use of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved in ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and increase the versatility of our knock-in system, we constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability.

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Crosstalk between translesion synthesis, Fanconi anemia network, and homologous recombination repair pathways in interstrand DNA crosslink repair and development of chemoresistance

Mutation Research/Reviews in Mutation Research ◽

10.1016/j.mrrev.2014.11.005 ◽

2015 ◽

Vol 763 ◽

pp. 258-266 ◽

Cited By ~ 31

Author(s):

Brittany Haynes ◽

Nadia Saadat ◽

Brian Myung ◽

Malathy P.V. Shekhar

Keyword(s):

Homologous Recombination ◽

Fanconi Anemia ◽

Translesion Synthesis ◽

Homologous Recombination Repair ◽

Recombination Repair ◽

Repair Pathways ◽

Crosslink Repair

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Disparate contributions of the Fanconi anemia pathway and homologous recombination in preventing spontaneous mutagenesis

Nucleic Acids Research ◽

10.1093/nar/gkm315 ◽

2007 ◽

Vol 35 (11) ◽

pp. 3733-3740 ◽

Cited By ~ 18

Author(s):

John M. Hinz ◽

Peter B. Nham ◽

Salustra S. Urbin ◽

Irene M. Jones ◽

Larry H. Thompson

Keyword(s):

Homologous Recombination ◽

Fanconi Anemia ◽

Spontaneous Mutagenesis ◽

Fanconi Anemia Pathway

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Fanconi Anemia FANCG Protein in Mitigating Radiation- and Enzyme-Induced DNA Double-Strand Breaks by Homologous Recombination in Vertebrate Cells

Molecular and Cellular Biology ◽

10.1128/mcb.23.15.5421-5430.2003 ◽

2003 ◽

Vol 23 (15) ◽

pp. 5421-5430 ◽

Cited By ~ 103

Author(s):

Kazuhiko Yamamoto ◽

Masamichi Ishiai ◽

Nobuko Matsushita ◽

Hiroshi Arakawa ◽

Jane E. Lamerdin ◽

...

Keyword(s):

Homologous Recombination ◽

Fanconi Anemia ◽

Cancer Susceptibility ◽

Bone Marrow Failure ◽

Chromosome Breakage ◽

Breast Cancer Susceptibility ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Brca1 And Brca2 ◽

Strand Breaks

ABSTRACT The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased ∼9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.

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Loss of the homologous recombination generad51leads to Fanconi anemia-like symptoms in zebrafish

10.1101/095646 ◽

2016 ◽

Author(s):

Jan Gregor Botthof ◽

Ewa Bielczyk-Maczyńska ◽

Lauren Ferreira ◽

Ana Cvejic

Keyword(s):

Bone Marrow ◽

Homologous Recombination ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Tumor Development ◽

Embryonic Stem ◽

Dominant Negative ◽

Hematopoietic Stem ◽

Recombination Protein

AbstractRAD51is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant negative mutations. FA is a hereditary DNA repair disorder characterized by various congenital abnormalities, progressive bone marrow failure and cancer predisposition. In this paper, we describe the first viable vertebrate model ofRAD51loss. Zebrafishrad51loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to crosslinking agents and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Co-mutation ofp53was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment ofRAD51as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this novel zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.Significance statementThe homologous recombination protein RAD51 has been extensively studied in prokaryotes and lower eukaryotes. However, there is a significant lack of knowledge of the role of this protein and its regulation in anin-vivocontext in vertebrates. Here we report the first viable vertebrate mutant model ofrad51in zebrafish. These mutant fish enabled us to confirm for the first time the recently discovered role ofRAD51in Fanconi anemia pathogenesis. We report that p53 linked embryonic stem cell defects directly lead to hematological impairments later in life. Co-mutation ofrad51withp53rescues the observed hematological defects, but predisposes the fish to early tumor development. The application of this model opens new possibilities to advance Fanconi anemia drug discovery.

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