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Updated: Jan 29, 2026

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
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Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Published on: February 17, 2011

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Evaluation of site-specific homologous recombination activity of BRCA1 by direct quantitation of gene editing

Yuki Yoshino1, Shino Endo1, Zhenghao Chen1

  • 1Department of Cancer Biology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryomachi, Aoba-ku, Sendai, 980-8575, Japan.

Scientific Reports
|February 9, 2019
PubMed

Insights

We developed the Assay for Site-specific HR Activity (ASHRA) to measure homologous recombination (HR) DNA repair. ASHRA accurately predicts cancer cell sensitivity to chemotherapy by evaluating HR activity.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cancer Research

Background:

  • Homologous recombination (HR) is crucial for repairing DNA double-strand breaks (DSBs) and inter-strand crosslinks.
  • HR activity in cancer cells correlates with sensitivity to DNA-damaging chemotherapeutic agents.
  • Accurate measurement of HR activity is vital for personalized cancer treatment and drug development.

Purpose of the Study:

  • To develop and validate a novel system, Assay for Site-specific HR Activity (ASHRA), for quantifying cellular HR activity.
  • To assess the utility of ASHRA in detecting HR deficiencies and predicting chemosensitivity.
  • To investigate the influence of specific genes and variants on HR activity in different genomic contexts.

Main Methods:

  • Transient transfection of cells with CRISPR/Cas9 and a HR donor vector containing a marker gene.
  • Induction of DSBs by Cas9, followed by repair via HR using the donor sequence.
  • Quantification of marker gene genomic integration using Western blotting, flow cytometry, or quantitative PCR (qPCR).

Main Results:

  • ASHRA successfully detected HR deficiency resulting from knockdown of BRCA1, BARD1, or RAD51, and from BRCA1 variants.
  • qPCR-based ASHRA results for BRCA1 variants correlated with the chemosensitivity of transfected cells.
  • ASHRA's qPCR format demonstrated the ability to measure HR activity in both transcribed and un-transcribed genomic regions, with no HR activity observed in transcriptionally inactive sites upon BRCA1 or BARD1 knockdown.

Conclusions:

  • ASHRA is a robust system for evaluating cellular HR activity.
  • ASHRA can predict patient sensitivity to chemotherapy, aiding in personalized treatment strategies.
  • The developed assay is valuable for screening HR-modulating drugs and advancing the understanding of HR mechanisms.

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