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Biology

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems

Published: February 2, 2024 doi: 10.3791/66501
1, 1, 1, 1, 1, 1
1Laboratory of Protein Structure and Function, Institute of Medicine and Pharmacy, Qiqihar Medical University

Abstract

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.

Introduction

A DNA double-strand break (DSB) is the most deleterious form of DNA damage, potentially leading to genome instability, chromosomal rearrangements, cellular senescence, and cell death if not repaired promptly1. Two well-established pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), are recognized for their effectiveness in addressing DNA DSBs2,3. HR is considered an error-free mechanism for DSB repair, utilizing homologous sequences in the sister chromatid as a template to restore the original configuration of the injured DNA molecule3. NHEJ, on the other hand, is an error-prone DSB repair pathway that joins the broken DNA ends without relying on any template2.

The NHEJ assay and HR assay are classical methods originally developed in Jasin's laboratory at Memorial Sloan-Kettering Cancer Center and utilized to quantify the efficiency of NHEJ and HR, respectively4,5,6,7. These assays play a crucial role in investigating the relevant mechanisms and factors associated with NHEJ and HR8,9,10,11,12,13,14. Both assays rely on the implementation of two disrupted GFP reporters, EJ5-GFP and DR-GFP, to monitor the repair of DSBs induced by the I-SceI endonuclease. The EJ5-GFP reporter is employed in the NHEJ assay, while the DR-GFP reporter is utilized in the HR assay. Each reporter is subtly designed so that the I-SceI-induced DSBs can only be repaired by a specific repair pathway to restore a GFP expression cassette4,5.

The NHEJ assay and HR assay can be conducted using either a chromosomally integrated or an extrachromosomal approach15,16. The chromosomally integrated approach necessitates the integration of the disrupted GFP reporters into the genome, allowing the analysis of DSB repair within a chromosomal context6,15. However, this approach requires prolonged cell passaging and is unsuitable for comparative studies involving multiple cell lines due to arbitrary chromosomal integration, introducing an additional confounding factor apart from inherent differences. In this protocol, we describe the extrachromosomal NHEJ assay and HR assays, involving the transient transfection of the disrupted GFP and I-SceI plasmids into HEK-293T cells, followed by flow cytometry analysis (the experiment workflow is shown in Figure 1). These non-integrated reporter assays were originally reported by Jasin's laboratory to study DNA interstrand cross-links repair16 and have been employed to assess NHEJ efficiency and HR efficiency by several laboratories9,10,11,12,13,14,17,18,19, including ours11. These extrachromosomal approaches facilitate the analysis of DSB repair in comparative studies involving multiple established stable cell lines.

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Protocol

1. Plasmid isolation

  1. Transform competent E. coli with the plasmids EJ5-GFP, DR-GFP, pCBASceI (I-SceI expressing plasmid), and PCI2-HA-mCherry (mCherry expressing plasmid) (see Table of Materials) following the standard transformation protocol20.
    NOTE: PCI2-HA-mCherry can be substituted with other mCherry or DsRed expressing plasmids.
  2. Cultivate the transformed E. coli in 500 mL of liquid LB medium supplemented with 100 µg/mL ampicillin overnight at 37 °C with shaking.
  3. Isolate the plasmids using a commercially available endotoxin-free plasmid maxi isolation kit following manufacturer's instructions (see Table of Materials).
  4. Quantify the plasmid concentrations using nanodrop microvolume spectrophotometers. Adjust the plasmid concentration to 1 µg/µL.

2. Cell preparation and transfection

  1. Culture HEK-293T cells in DMEM medium supplemented with 10% fetal bovine serum. Ensure that the HEK-293T cells or established HEK-293T stable cells are in good growth status.
    NOTE: Frozen cells should undergo at least two rounds of splitting before transfection.
  2. On the day prior to transfection, seed HEK-293T cells in a 6-well plate at 2 × 105 cells/well to achieve around 60% confluency the following day.
  3. On the day of transfection, confirm the cell confluency, aiming for approximately 60%. For the NHEJ assay, transfect the experimental sample cells in a 6-well plate with 1 µg of EJ5-GFP, 1 µg of pCBASceI, and 1 µg of PCI2-HA-mCherry plasmids, using a commercial transfection reagent following the manufacturer's instructions (see Table of Materials). For the HR assay, replace the EJ5-GFP plasmid with the DR-GFP plasmid.
  4. For FACS calibration controls, leave a well of cells untransfected as a negative control. Transfect the cells in a 6-well plate with either (1) 1 µg of EJ5-GFP (for NHEJ assay) or 1 µg of DR-GFP (for HR assay), together with 1 µg of pCBASceI as a GFP single-color control, or (2) 1 µg of PCI2-HA-mCherry as an mCherry single-color control.

3. Analysis of the GFP-positive and mCherry-positive cells by flow cytometry

  1. Two days after transfection, confirm the expression of GFP and mCherry proteins using a fluorescent microscope.
  2. Three days after transfection, remove the media from the wells, wash once with PBS, and add 500 µL trypsin to each well. Incubate for 5 min at 37 °C to detach all cells from the plate. Throughout this and the subsequent steps, shield the cells from direct light.
  3. Add 500 µL complete DMEM media to each well, resuspend cells, ensuring no clumps are visible.
  4. Transfer all cells from each well into 1.5 mL centrifuge tubes, then centrifuge cells for 2 min at 1000 × g at room temperature.
  5. Carefully remove the supernatant by pipetting and wash once with 1.0 mL PBS.
  6. Again, carefully remove the supernatant by pipetting, resuspend the cells in 500 µL of PBS, and transfer the cells to FACS tubes (see Table of Materials).
    NOTE: For optimal results, commence flow cytometry analysis immediately after cell harvest. Alternatively, cells can be stored on ice for several hours.
  7. Calibrate the FACS with untransfected cells (negative control), EJ5-GFP/DR-GFP, and pCBASceI-transfected cells (GFP single-color control) and mCherry-transfected cells (mCherry single-color control). Adjust voltage and compensation to minimize fluorescence spillover between GFP and mCherry.
  8. Acquire and record at least 10,000 intact cells from each tube.

4. Data analysis

NOTE: The following steps are performed using FlowJo software (see Table of Materials) to analyze the FACS Data. Comparable procedures can be executed in alternative analysis software.

  1. Drag all FACS files into the dashboard. Double-click on one of the files to display the FSC/SSC plot.
  2. Create a polygon gate to select intact cells, excluding debris in the lower-left corner. Name this subpopulation "Intact Cells" and drag this gate to the "All Samples" bar. Scroll through each sample using the Next Sample button to ensure appropriate gating for each sample (Figure 2A).
  3. Double-click on the Intact Cells subpopulation of the negative control sample (untransfected cells) to bring up a new plot. Change the axes to FL1-H (x-axis, GFP) and FL2-H (y-axis, mCherry).
  4. Select the Quad Gating tool and click on the upper-right extreme of the negative cell population (Figure 2B). Drag the four rectangular gates to the "Intact Cells" bar. Scroll through samples of GFP single-color control or mCherry single-color control using the Next Sample button to ensure appropriate gating for discriminating GFP-positive or mCherry-positive from negative control cells (Figure 2C,D). Fine-tune the quadrant gating if necessary and apply this gating to all "Intact Cells" populations.
  5. Click on the Layout Editor. Right-click on the representative plots and choose Copy to Layout Editor. Select all the representative plots, then double-click to open Graph Definition. Adjust the axis label, annotate, and legend as desired.
  6. The percentage of mCherry-positive cells in experimental samples serves as the transfection efficiency control. Calculate the relative DNA repair efficiency by comparing the number of GFP-positive cells with the number of mCherry-positive cells in the same plot.

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Representative Results

To ensure the accuracy of NHEJ and HR analysis, the implementation of a suitable compensation adjustment and gating strategy is necessary. Typically, mCherry fluorescence does not manifest in the GFP detector when using a 530 nm filter. However, in instances of cells exhibiting extremely high GFP expression, the GFP fluorescence may contaminate the mCherry detector when using a 575 nm filter. To address these concerns, negative control, GFP single-color control, and mCherry single-color control samples were used for compensation and to regulate the gating strategy. Typical FACS results of the control cells are shown in Figure 2. Compensation adjustment and gating strategy should position the majority of GFP-positive cells in the lower-right quadrant (Figure 2C) and the majority of mCherry-positive cells in the upper-left quadrant (Figure 2D).

Final representative plots are shown in Figure 3A,B. Following the induction of DSBs by I-SceI, successful repair events will reconstitute the GFP gene. Hence, the percentage of GFP-positive cells corresponds to the efficiency of DNA DSB repair and transfection efficiency. The percentage of mCherry-positive cells only corresponds to the transfection efficiency. The relative efficiency of DNA DSB repair can be calculated as the ratio of GFP-positive cells to mCherry-positive cells (Figure 3C).

A concrete example of the NHEJ assay to characterize the role of Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) in DNA repair is shown in Figure 4. In this example, we performed the NHEJ assay to assess the NHEJ efficiency of shControl cells and shWASH cells (Figure 4). Obviously, the loss of WASH resulted in a reduction in NHEJ efficiency, confirming the role of WASH in promoting NHEJ efficiency as determined by the NHEJ assay.

Figure 1
Figure 1: Overview of the workflow for NHEJ/HR assay. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Gating strategy for flow cytometry analysis. (A) FSC-H/SSC-H gating on intact cells. (B) Untransfected HEK-293T cells were used as a negative control for excluding autofluorescent cells. (C) HEK-293T cells transfected with EJ5-GFP and pCBASceI plasmids were used for setting the gating for GFP-positive cells. (D) HEK-293T cells transfected with mCherry plasmids were used for setting the gating for mCherry-positive cells. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Typical results. (A) Representative plots illustrating the analysis of NHEJ. (B) Representative plots illustrating the analysis of HR. (C) Model diagram of calculation method. Please click here to view a larger version of this figure.

Figure 4
Figure 4: WASH promotes NHEJ efficiency. (A) Representative plots illustrating the analysis of NHEJ efficiency in shControl cells and shWASH cells. (B) Representative statistical results comparing NHEJ efficiency between shControl and shWASH cells. The data is presented as mean ± SD. Please click here to view a larger version of this figure.

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Discussion

The method described here has been employed in several papers to assess NHEJ efficiency and HR efficiency9,10,11,12,13,14,16,17,18,19. This method is pertinent for elucidating the underlying mechanisms of DNA DSB repair and identifying new factors associated with NHEJ and HR. For instance, the function of DNA-dependent protein kinase catalytic subunit (DNA-PKcs)9, Coilin-interacting nuclear ATPase protein (hCINAP)10, WASH11, retinoic acid-inducible gene I (RIG-I)12, X-ray repair cross-complementing protein 4 (XRCC4)12, HORMA domain-containing protein 1 (HORMAD1)13, ubiquitin-specific protease 44 (USP44)14, and lncRNA NIHCOLE (noncoding RNA induced in hepatocellular carcinoma with an oncogenic role in ligation efficiency) in NHEJ or HR have been unveiled or validated through the application of extrachromosomal methods. In contrast to the chromosomally integrated approach, this extrachromosomal approach enables swift analysis of NHEJ or HR efficiency through the utilization of established stable cell lines.

The extrachromosomal NHEJ and HR assays, which have been previously utilized in HEK-293T cells by us and others10,11,12, have also been employed by several other research groups in various cell lines, including U2OS cells16,17, embryonal carcinoma cells17,19, HCT116 cells9, SUM159 cells13, HCC1143 cells13, MDA-MB-436 cells13, SUNE1 cells14, Huh7 cells18, and JHH6 cells18. The protocol here provides a detailed description of the NHEJ and HR assays conducted in HEK-293T cells. Given the notable transfection efficiency of HEK-293 cells, a combination of 1 µg of EJ5-GFP or 1 µg DR-GFP, along with 1 µg pCBASceI, is deemed adequate for HEK-293T cells cultured in a 6-well plate. However, the transfection conditions may differ in cell lines other than HEK-293T9,16,17, necessitating the investigator to meticulously determine the exact conditions for successful transfection.

Attaining the requisite cell confluency prior to transfection is a pivotal procedure in the protocol. An insufficient or excessive number of cells can substantially impede the efficacy of transfection. An inadequate number of cells exacerbates post-transfection toxicity, thereby impeding the precise evaluation of DSB repair. Furthermore, it is important to use the same mix of plasmids and transfection reagent within one experiment, as deviations in the mixture of EJ5-GFP/DR-GFP, pCBASceI, PCI2-HA-mCherry, and transfection reagent may potentially impact the result.

In addition to NHEJ and HR, alternate end joining (a-EJ) and single-strand annealing (SSA) have been identified as additional mechanisms for repairing DSBs. However, in wild-type cells, NHEJ and HR are the primary pathways for DSB repair, while a-EJ and SSA only make a minor contribution in this regard. It's worth noting that the NHEJ assay utilizing EJ5-GFP plasmid detects both NHEJ and a-EJ events, and thus can be considered as an assay for total-NHEJ, including both NHEJ and a-EJ5. An individual analysis of a-EJ can be simply achieved by replacing the EJ5-GFP with EJ2-GFP18. Similar assays could also be performed to analyze the repair efficiency of SSA by replacing the disrupted GFP reporter with hprtSAGFP21.

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Disclosures

There are no conflicts of interest to disclose.

Acknowledgments

This research was funded by the Natural Science Foundation of Heilongjiang Province of China (YQ2022C036) and the Graduate Innovation Foundation of Qiqihar Medical University (QYYCX2022-06). Figure 1 produced using MedPeer.

Materials

Name Company Catalog Number Comments
6 cm dishes  BBI F611202-9001
6 well plates Corning 3516
Ampicillin Beyotime ST007 Working concentration: 100 μg/mL
DH5α Competent Cells TIANGEN CB101
DMEM Hyclone SH30022.01
DR-GFP Addgene 26475
EJ5-GFP Addgene 44026
EndoFree Maxi Plasmid  kit TIANGEN DP117 alternative endotoxin-free plasmid extraction kit can be used
FACS tubes FALCON 352054
Fetal bovine serum CLARK FB25015
Flow cytometer BD Biosciences BD FACSCalibur
FlowJo V.10.1 Treestar alternative analysis software can be used
HEK-293T cells National Infrastructure of Cell Line Resource 1101HUM-PUMC000091
Lipo3000 Invitrogen L3000015 alternative transfection regents can be used
PBS Biosharp BL601A
pCBASceI Addgene 26477 I-SceI expressing plasmid
PCI2-HA-mCherry alternative plasmids containing DsRed can be used
Trypsin Gibco 25200-056

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References

  1. Huang, R., Zhou, P. K. DNA damage repair: historical perspectives, mechanistic pathways and clinical translation for targeted cancer therapy. Signal Transduct Target Ther. 6 (1), 254 (2021).
  2. Pannunzio, N. R., Watanabe, G., Lieber, M. R. Nonhomologous DNA end-joining for repair of DNA double-strand breaks. J Biol Chem. 293 (27), 10512-10523 (2018).
  3. Wright, W. D., Shah, S. S., Heyer, W. -D. Homologous recombination and the repair of DNA double-strand breaks. J Biol Chem. 293 (27), 10524-10535 (2018).
  4. Pierce, A. J., Johnson, R. D., Thompson, L. H., Jasin, M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 13 (20), 2633-2638 (1999).
  5. Bennardo, N., Cheng, A., Huang, N., Stark, J. M. Alternative-NHEJ Is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4 (6), e1000110 (2008).
  6. Gunn, A., Stark, J. M. I-SceI-based assays to examine distinct repair outcomes of mammalian chromosomal double strand breaks. Methods Mol Biol. 920, 379-391 (2012).
  7. Zuo, N., et al. Detection of alternative end-joining in HNSC cell lines using DNA Double-strand break reporter assays. Bio Protoc. 12 (17), 4506 (2022).
  8. Schrank, B. R., et al. Nuclear ARP2/3 drives DNA break clustering for homology-directed repair. Nature. 559 (7712), 61-66 (2018).
  9. Lu, H., Saha, J., Beckmann, P. J., Hendrickson, E. A., Davis, A. J. DNA-PKcs promotes chromatin decondensation to facilitate initiation of the DNA damage response. Nucleic Acids Res. 47 (18), 9467-9479 (2019).
  10. Xu, R., et al. hCINAP regulates the DNA-damage response and mediates the resistance of acute myelocytic leukemia cells to therapy. Nat Commun. 10 (1), 3812 (2019).
  11. Wang, T., et al. WASH interacts with Ku to regulate DNA double-stranded break repair. iScience. 25 (1), 103676 (2022).
  12. Guo, G., et al. Reciprocal regulation of RIG-I and XRCC4 connects DNA repair with RIG-I immune signaling. Nat Commun. 12 (1), 2187 (2021).
  13. Watkins, J., et al. Genomic complexity profiling reveals that HORMAD1 overexpression contributes to homologous recombination deficiency in triple-negative breast cancers. Cancer Discov. 5 (5), 488-505 (2015).
  14. Chen, Y., et al. USP44 regulates irradiation-induced DNA double-strand break repair and suppresses tumorigenesis in nasopharyngeal carcinoma. Nat Commun. 13 (1), 501 (2022).
  15. Seluanov, A., Mao, Z., Gorbunova, V. Analysis of DNA double-strand break (DSB) repair in mammalian cells. JoVE. (43), e2002 (2010).
  16. Nakanishi, K., Cavallo, F., Brunet, E., Jasin, M. DNA recombination: Methods and Protocols. Tsubouchi, H. , Humana Press. Totowa, NJ. 283-291 (2011).
  17. Cavallo, F., et al. Reduced proficiency in homologous recombination underlies the high sensitivity of embryonal carcinoma testicular germ cell tumors to cisplatin and poly (ADP-Ribose) polymerase inhibition. PLoS One. 7 (12), e51563 (2012).
  18. Unfried, J. P., et al. Long noncoding RNA NIHCOLE promotes ligation efficiency of DNA double-strand breaks in hepatocellular carcinoma. Cancer Res. 81 (19), 4910-4925 (2021).
  19. Cavallo, F., Caggiano, C., Jasin, M., Barchi, M. Testicular Germ Cell Tumors: Methods and Protocols. Bagrodia, A., Amatruda, J. F. , Springer US. New York, NY. 113-123 (2021).
  20. Green, M. R., Sambrook, J. The Hanahan method for preparation and transformation of competent Escherichia coli: high-efficiency transformation. Cold Spring Harbor Protocols. 2018 (3), 101188 (2018).
  21. Stark, J. M., Pierce, A. J., Oh, J., Pastink, A., Jasin, M. Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol Cell Biol. 24 (21), 9305-9316 (2004).
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Cite this Article

Zhang, L. P., Nie, Y. H., Tang, T.,… More

Zhang, L. P., Nie, Y. H., Tang, T., Zheng, A. X., Hong, X., Wang, T. Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems. J. Vis. Exp. (204), e66501, doi:10.3791/66501 (2024).

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