Background

Homologous recombination (HR) is an essential pathway to repair DNA double-strand breaks (DSBs) and contributes to the repair of interstrand crosslinks. Germline pathogenic variants of HR-related genes, such as BRCA1 and BRCA2 (BRCA1/2), cause hereditary breast and ovarian cancer syndrome (HBOC) [1]. Sporadic cancers also have alterations of HR-related genes [2]. Cancer cells with HR deficiency are sensitive to cancer treatments, including poly(ADP-ribose) polymerase (PARP) inhibitors, DNA-damaging agents such as platinum agents, and ionizing radiation [3,4]. Thus, accurate evaluation of HR activity is helpful to diagnose HBOC and stratify patients for cancer therapy.

For diagnosis of HBOC and stratification of patients for treatment with PARP inhibitors, genetic tests are performed to detect pathogenic variants of BRCA1/2 and other HR-related genes in the clinic. However, variants of uncertain significance have been identified, and an accurate functional assay to evaluate HR activity is required for appropriate clinical decision-making [5]. In addition, genomic scarring assays are used to detect HR deficiency in tumor tissues and predict the effects of PARP inhibitors [6] but do not detect changes in HR activity caused by revertant mutations of HR-related genes.

To evaluate cellular HR activity, the direct-repeat GFP (DR-GFP) assay has been utilized [7,8]. However, this assay targets the exogenous GFP locus and detects HR products at the protein level to evaluate HR activity, and its results tend to be qualitative [9]. In addition, the DR-GFP assay requires the establishment of a stable cell line [7,8]. DSB repair can be evaluated by immunostaining for RAD51 (an essential HR factor) or g-H2AX (a DSB marker). However, evaluation of RAD51 foci does not reflect HR activity due to alteration of steps downstream of RAD51 focus formation, and DSBs detected by g-H2AX foci are repaired by non-homologous end-joining in addition to HR [10,11]

To overcome these limitations, we developed an assay to evaluate HR activity called Assay for Site-Specific HR Activity (ASHRA) (Figure 1) [12]. In ASHRA, we use Cas9 endonuclease to create DSBs at specific sites in the genome, enabling the targeting of any endogenous loci. The marker sequence in the donor vector is integrated into the genome during the repair of the DSB by HR. The efficiency of integration of the marker sequence is evaluated by real-time PCR.

Quantification of HR products at the DNA level allows the method to be quantitative [9]. Furthermore, ASHRA can be performed by transiently transfecting two plasmids and does not require the establishment of a stable cell line. We successfully evaluated HR activity in lymphocyte-derived cells and tumor tissues by ASHRA using electroporation for plasmid delivery [13]. The direct measurement of HR activity in tumor tissues accurately predicted sensitivity to PARP inhibitors in vivo [13]. Thus, ASHRA can be performed using a wide range of samples, which greatly widens the application of HR activity evaluation.

Collectively, ASHRA allows easy and quantitative evaluation of HR activity at any locus of interest in the genome. Using ASHRA, we can directly evaluate HR activity in various samples irrespective of the causal genes, type of sequence variations, and shortage of functional information about the variation. In this article, we present the protocols of ASHRA for the measurement of HR activity in adherent cells, suspension cells, and tumor tissues.