Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

1. Preparation and quality control (QC) of reporter substrates

NOTE: The plasmids encoding the reporter substrates can be propagated in standard Escherichia coli strains (e.g., DH5α and derivates) and recovered by plasmid isolation. Plasmid details (size and antibiotic resistance) are described in Table 1 and Figure 1.

Figure 1: Schematics of extrachromosomal NanoLuc-based DSBR reporter assays. Schematic representation of DSBR reporter substrates, including their location within each source plasmid, main sequence features, and final layout after pathway-specific repair. The resection-independent MMEJ substrate core is excised from the source plasmid by digestion with XhoI/HindIII, after which caps must be ligated to both ends to produce the substrate for MMEJ. The blunt NHEJ reporter substrate is generated by excision from the source plasmid with EcoRV. The resection-dependent MMEJ, non-blunt NHEJ, long template HR, and SSA reporter substrates are generated by linearization of the source plasmids using either I-SceI (which produces non-cohesive ends) or HindIII (which produces cohesive ends). The short template HR reporter substrate is generated by linearization of the source plasmid using I-SceI (which produces cohesive ends). Repair of each reporter substrate by the target DSBR pathway reconstitutes an intact nanoluciferase ORF encoding functional NanoLuc. This figure was adapted from Rajendra et al.⁷. Abbreviations: DSBR = double strand break repair; NanoLuc = Nanoluciferase; MMEJ = microhomology-mediated end joining; NHEJ = non-homologous end joining; HR = homologous recombination; SSA = single strand annealing; ORF = open reading frame. Please click here to view a larger version of this figure.

1. Preparation of resection-independent MMEJ reporter substrate (Figure 1 and Figure 2A-C)
   1. Preparation of ssDNA/dsDNA caps (Figure 2A)
      1. Resuspend the four oligonucleotides listed in Table 2 individually in annealing buffer (20 mM Tris pH 7.5, 50 mM NaCl in molecular biology grade water) to generate a stock solution at 100 µM.
      2. Annealing of ss/dsDNA caps
         1. Mix 50 µL each of the long and short oligonucleotides (100 µM stock solution) for the left cap from step 1.1.1.1 in a 0.2 mL tube.
         2. Repeat for right cap oligonucleotides.
         3. Using a thermocycler, incubate each oligonucleotide mix at 99 °C for 5 min then ramp down to 10 °C at 1 °C/min.
            NOTE: This will produce the annealed left and right caps.
      3. (QC, optional) Verification of oligonucleotide annealing by electrophoresis
         1. Electrophorese 100 ng of each product from step 1.1.1.2 alongside individual oligonucleotides from step 1.1.1.1 and a low molecular weight DNA ladder in a 20% acrylamide-Tris-Borate-EDTA (TBE) gel at 200 V for 80 min.
         2. Stain the gel with TBE running buffer containing an appropriate fluorescent DNA dye for visualization of both ssDNA and dsDNA for at least 10-15 min.
         3. Visualize fluorescence on a gel documentation system (Figure 2A).
            NOTE: Steps 1.1.1.3.1 to 1.1.1.3.3 are used to verify that caps have correctly annealed. The apparent sizes for the left cap and right cap should be ~120 bp and ~175 bp, respectively.
   2. Purification of reporter substrate core (Figure 2B)
      1. Mix 100 µg of the reporter core plasmid with 4 µL of HindIII and 4 µL of XhoI (4 U/µg plasmid for each enzyme) and 20 µL of 10x buffer. Bring the total volume to 200 µL with double-distilled water (ddH₂O) and incubate at 37 °C for 2 h to overnight to digest plasmid.
         NOTE: For digestion of larger or smaller quantities, scale the reaction proportionally.
      2. Incubate digestion reaction at 80 °C for 20 min to heat inactivate restriction enzymes.
      3. Add 40 µL of alkaline phosphatase (2 U/µg plasmid) and 27 µL of 10x buffer to the reaction mix from step 1.1.2.2. Incubate at 37 °C for 2 h to dephosphorylate the plasmid.
         NOTE: Scale reaction up or down as needed.
      4. Add 60 µL of 6x loading dye to the reaction from step 1.1.2.3. Electrophorese alongside a high molecular weight DNA ladder in a 1.5 % (w/v) agarose-Tris-Acetate-EDTA (TAE) gel containing a fluorescent dsDNA stain at 120 V for 2.5 h or until bands are sufficiently resolved.
      5. Visualize fluorescence on a gel documentation system (Figure 2B).
      6. Excise the reporter core fragment (~1.5 kb) from the gel using a clean scalpel, taking care not to contaminate with vector backbone (~2.5 kb).
      7. Extract the reporter core fragment using a gel extraction kit, following the manufacturer's instructions.
      8. Measure the DNA concentration and quality (A_260/280) by spectrophotometry.
   3. Ligation of reporter substrate core with ssDNA/dsDNA caps and purification of final reporter substrate (Figure 2C)
      1. Mix 30 µg of the reporter core fragment from step 1.1.2.7 with 3.85 µL of each annealed left and right cap from step 1.1.2 (approx. 6:1 molar ratio of cap:core DNA), 30 µL of 10x ligase buffer, and 1.5 µL of T4 DNA ligase (20 U/µg DNA). Bring the total volume of the reaction to 300 µL with ddH₂O and incubate overnight at 16 °C to ligate caps to reporter core fragment.
         NOTE: Scale reaction up or down as required.
      2. (QC, optional) Electrophorese 200 ng of the resulting product from step 1.1.3.1 alongside the reporter substrate core and a high molecular weight DNA ladder in a 0.7% (w/v) agarose-TAE gel containing a fluorescent dsDNA stain at 120 V for at least 1.5 h. Verify that the band corresponding to the ligated product migrates at a slightly slower rate than the non-ligated reporter substrate core (Figure 2C).
         NOTE: This QC step is to verify that the ligation of caps to the reporter core fragment has taken place correctly.
      3. Purify the DNA from the digestion reaction in step 1.3.1. using the preferred method (e.g., a bead-based or column-based method), following the manufacturer's instructions.
         NOTE: The purification step 1.1.3.3 substantially reduces the presence of unligated caps.
   4. Measure DNA concentration and quality (A_260/280) by spectrophotometry.
2. Preparation of blunt NHEJ reporter substrate (Figure 1 and Figure 2D,E)
   1. Mix 100 µg of the reporter core plasmid with 5 µL of EcoRV (5 U/µg plasmid) and 20 µL of 10x buffer. Bring the total volume to 200 µL with ddH₂O and incubate at 37 °C for 2 h to overnight to digest plasmid.
      NOTE: For digestion of larger or smaller quantities, scale the reaction proportionally.
   2. Incubate the digestion reaction at 65 °C for 20 min to heat inactivate restriction enzyme.
   3. Add 50 µL of 6x loading dye to the reaction from step 1.2.2. Electrophorese alongside a high molecular weight DNA ladder in a 1.5% (w/v) agarose-TAE gel containing a fluorescent dsDNA stain at 120 V for 2 h or until bands are sufficiently resolved.
   4. Visualize fluorescence on a gel documentation system (Figure 2D).
   5. Excise the reporter substrate (~1.7 kb) from the gel using a clean scalpel, taking care not to contaminate with the vector backbone (~2.6 kb).
   6. Extract the reporter core fragment using a gel extraction kit, following the manufacturer's instructions (Figure 2E).
   7. Measure the DNA concentration and quality (A_260/280) by spectrophotometry.
3. Preparation of I-SceI-based reporter substrates (Figure 1)
   NOTE: These substrates include resection-dependent MMEJ (Figure 2F), non-blunt NHEJ (Figure 2G), long template HR (Figure 2H), short template HR (Figure 2I), and SSA (Figure 2J).
   1. Mix 100 µg of the reporter core plasmid with 50 µL of I-SceI (5 U/µg plasmid) and 60 µL of 10x buffer. Bring the total volume to 600 µL with ddH₂O and incubate at 37 °C for 2 h to overnight to digest plasmid.
      NOTE: For digestion of larger or smaller quantities, scale the reaction proportionally. Non-blunt NHEJ, resection-dependent MMEJ, long template HR and SSA plasmids can also be digested with HindIII, which will generate complementary cohesive ends.
   2. Incubate the digestion reaction at 65 °C for 20 min to heat inactivate I-SceI. Set the temperature to 80 °C during this step if HindIII was used for digestion instead.
   3. Purify DNA from the inactivated digestion reaction in step 1.3.2 using the preferred method (e.g., a bead-based or column-based method), following the manufacturer's instructions.
   4. Measure the DNA concentration and purity A_260/280) by spectrophotometry.
4. Quality control of reporter substrates
   1. Electrophorese 200 ng of the reporter substrate alongside a high molecular weight DNA ladder in a 0.7% (w/v) agarose-TAE gel containing a fluorescent dsDNA stain at 120 V for 2 h or until bands are sufficiently resolved. Load the original uncut reporter plasmid alongside as a control.
   2. Verify that the defined bands of the correct size are observed for each reporter substrate (refer to Table 1 and Figure 2F-J).

Figure 2: Gel electrophoretic analyses of DSBR reporter substrate generation. (A-C) Representative images from gel electrophoretic analysis of intermediates and final construct required for the generation of the resection-independent MMEJ reporter substrate. The adjacent images in panels (A) and (C) are from the same gels; irrelevant lanes have been omitted. (D,E) Representative images from gel electrophoretic analysis for source plasmid, EcoRV-digested products, and gel-extracted final construct required for the generation of the blunt NHEJ reporter substrate. (F-J) Representative images from gel electrophoretic analysis of source plasmids and linearised DSBR constructs for the I-SceI-digested reporter substrates: resection-dependent MMEJ, non-blunt NHEJ, long template HR, short template HR, SSA. Abbreviations: DSBR = double strand break repair; MMEJ = microhomology-mediated end joining; NHEJ = non-homologous end joining; HR = homologous recombination; SSA = single strand annealing. Please click here to view a larger version of this figure.

2. Transient transfection of DSBR reporter substrates

NOTE: An overview of the experimental workflow of assays and some potential permutations are outlined in Figure 3. The protocol below describes an experiment using HEK-293 cells, where these are reverse-transfected with the reporter substrate. Numbers below can be scaled up or down according to the number of wells to be used per plate. The following steps are calculated for an assay performed in one 96-well plate; see Discussion for additional considerations.

Figure 3: Experimental workflow for DSBR reporter assays. Cells of interest are transfected with the linearised DSBR substrate (coding for a NanoLuc ORF that needs to be repaired through a specific DNA repair event) and a Firefly plasmid (transfection control). Then, 6-24 h post transfection, the Firefly luminescence and NanoLuc luminescence can be read sequentially following the addition of Nano-Glo Dual-Luciferase reagents. Example permutations to the core steps (shown within light blue boxes) can be used to test how genetic modulation (knockout, knockdown, or overexpression) or pharmacological treatment affects DSBR pathway proficiency in cells. Abbreviations: DSBR = double strand break repair; NanoLuc = Nanoluciferase; WT = wild type; KO = knockout. Please click here to view a larger version of this figure.

1. (Optional) If assessing the impact of compounds on the reporter assay, dispense compounds dissolved in vehicle (e.g., dimethyl sulfoxide [DMSO]) into wells of a 96-well plate according to a predefined experimental layout. Normalize vehicle concentration across all wells.
   NOTE: Technical replicates are recommended. Include control wells (e.g., vehicle-only).
2. In a 1.5 mL tube, dilute Firefly control plasmid (control luciferase) and NanoLuc DSBR reporter substrate (reporter luciferase) generated in steps 1.1, 1.2 or 1.3, in 500 µL of transfection buffer. Use 0.66 µg of control luciferase plasmid per 1 × 10⁶ cells. See Table 3 for the quantities of NanoLuc DSBR reporter substrate to use.
   NOTE: A positive control plasmid that constitutively expresses the reporter luciferase can be used to validate instrument setup and transfection conditions or to check for non-specific effects on the reporter luciferase itself. As a starting point, we recommend 0.1 µg of reporter luciferase plasmid and 0.66 µg of control luciferase plasmid per 1 × 10⁶ cells.
3. Add lipid-based transfection reagent to the diluted DNA from step 2.2 at a manufacturer's recommended ratio (e.g., 1:2, µg DNA:µL reagent for the reagent described in the Table of Materials), mix well by vortexing briefly, and incubate for 10 min at room temperature.
4. Harvest cells by trypsinization, resuspend them in fresh medium containing 10% fetal bovine serum (FBS), and count them.
5. Transfer 3 × 10⁶ cells into a 15 mL tube and resuspend in 8.5 mL of medium.
6. Add the DNA transfection mix from step 2.3 into the cell suspension and mix several times by inversion.
7. Plate 80 µL of cell suspension (approximately 2.7 × 10⁴ cells) per well.
   NOTE: The suspension containing cells and DNA transfection mix can be dispensed onto the plate either by pipetting manually or using an automated liquid handler for higher-throughput experiments.
8. Incubate at 37 °C/5% CO₂ for 24 h.

3. Detection of luminescence

1. Preparation of reagents
   1. Follow the manufacturer's instructions for the preparation and addition of reporter (NanoLuc) and control (Firefly) luciferase reagents, which provide the substrates for both luciferases (Furimazine and 5´-Fluoroluciferin, respectively):
   2. Prepare control luciferase reagent. Reconstitute according to the manufacturer's instructions and mix by inversion until the luciferase substrate is thoroughly dissolved.
   3. Prepare fresh reporter luciferase reagent according to the manufacturer's instructions. Calculate the amount of reagent needed to add 80 µL/well and add substrate into an appropriate volume of assay buffer at a 1:100 ratio (luciferase substrate:buffer). For one 96-well plate, dilute 88 µL of luciferase substrate into 8,800 µL of buffer and mix by inversion.
      NOTE: These quantities include an approximate 10% excess. Once reconstituted, the control luciferase reagent can be stored according to the manufacturer's instructions for future use. The reporter luciferase reagent must be prepared fresh for each use.
2. Serial detection of luminescence from control and reporter luciferases (96-well plate)
   1. Allow the plate and luciferase reagents to reach room temperature.
   2. Add 80 µL of control luciferase reagent per well.
   3. Shake the plate for 3 min on an orbital shaker at 450 rpm.
   4. Measure the control luciferase luminescence signal with a luminescence plate reader.
      NOTE: Read emission at 580 nm (80 nm bandpass filter) or the total luminescence per well. This luminescence is used as a measure of transfection efficiency and cell density and can also inform about cellular toxicity caused by the test treatments.
   5. Add 80 µL of reporter luciferase reagent per well.
      NOTE: This reagent will inhibit the control luciferase; it also contains the substrate for the reporter luciferase.
   6. Shake the plate for 3 min on an orbital shaker at 450 rpm.
   7. Leave the plate to rest for 7 min at room temperature.
   8. Measure the reporter luciferase luminescence signal with a luminescence plate reader.
      NOTE: Read emission at 470 nm (80 nm bandpass filter) or, alternatively, total luminescence per well. This luminescence gives information on the amount of reporter substrate that has been repaired by the DSBR pathway of interest.
   9. Export luminescence readings for downstream analysis.

4. Data analysis

1. Divide the reporter luciferase (NanoLuc) luminescence signal by the control luciferase (Firefly) luminescence signal originating from the same well to calculate the assay signal. Apply that calculation to all the wells as shown in equation (1).
        (1)
2. Calculate the average of the assay signals for the control wells (e.g., vehicle-only).
3. Normalize the assay signals for test wells to the control well average using equation (2) to calculate Repair (%) per well.
   (2)
4. (Optional, curve fitting) If testing multiple compound doses, plot the calculated Repair (%) against the compound concentration and fit a dose-response curve using a non-linear regression model. Use this curve for subsequent EC₅₀ interpolation.