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.
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.
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.
1. Plasmid isolation
2. Cell preparation and transfection
3. Analysis of the GFP-positive and mCherry-positive cells by flow cytometry
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.
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: Overview of the workflow for NHEJ/HR assay. Please click here to view a larger version of this figure.
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: 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: 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.
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.
The authors have nothing to disclose.
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.
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 |