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

Summary

This protocol describes an extrachromosomal nonhomologous end joining (NHEJ) assay and homologous recombination (HR) assay to quantify the efficiency of NHEJ and HR in HEK-293T cells.

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 DSBs^2,3. 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, respectively^4,5,6,7. These assays play a crucial role in investigating the relevant mechanisms and factors associated with NHEJ and HR^{8,9,10,11,12,13,14}. 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 cassette^4,5.

The NHEJ assay and HR assay can be conducted using either a chromosomally integrated or an extrachromosomal approach^15,16. The chromosomally integrated approach necessitates the integration of the disrupted GFP reporters into the genome, allowing the analysis of DSB repair within a chromosomal context^6,15. 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 laboratories^{9,10,11,12,13,14,17,18,19}, including ours¹¹. These extrachromosomal approaches facilitate the analysis of DSB repair in comparative studies involving multiple established stable cell lines.

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.

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 comp...

Discussion

The method described here has been employed in several papers to assess NHEJ efficiency and HR efficiency^{9,10,11,12,13,14,16,17,18,19}. This method is pertinent for elucidating the underly...

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