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.

1. Plasmid isolation
<ol>
	<li>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.</li>
	<li>Cultivate the transformed E. coli in 500 mL of liquid LB medium supplemented with 100 &#181;g/mL ampicillin overnight at 37 &#176;C with shaking.</li>
	<li>Isolate the plasmids using a commercially available endotoxin-free plasmid maxi isolation kit following manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Quantify the plasmid concentrations using nanodrop microvolume spectrophotometers. Adjust the plasmid concentration to 1 &#181;g/&#181;L.</li>
</ol>
2. Cell preparation and transfection
<ol>
	<li>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.</li>
	<li>On the day prior to transfection, seed HEK-293T cells in a 6-well plate at 2 &#215; 105 cells/well to achieve around 60% confluency the following day.</li>
	<li>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 &#181;g of EJ5-GFP, 1 &#181;g of pCBASceI, and 1 &#181;g of PCI2-HA-mCherry plasmids, using a commercial transfection reagent following the manufacturer&#39;s instructions (see Table of Materials). For the HR assay, replace the EJ5-GFP plasmid with the DR-GFP plasmid.</li>
	<li>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 &#181;g of EJ5-GFP (for NHEJ assay) or 1 &#181;g of DR-GFP (for HR assay), together with 1 &#181;g of pCBASceI as a GFP single-color control, or (2) 1 &#181;g of PCI2-HA-mCherry as an mCherry single-color control.</li>
</ol>
3. Analysis of the GFP-positive and mCherry-positive cells by flow cytometry
<ol>
	<li>Two days after transfection, confirm the expression of GFP and mCherry proteins using a fluorescent microscope.</li>
	<li>Three days after transfection, remove the media from the wells, wash once with PBS, and add 500 &#181;L trypsin to each well. Incubate for 5 min at 37 &#176;C to detach all cells from the plate. Throughout this and the subsequent steps, shield the cells from direct light.</li>
	<li>Add 500 &#181;L complete DMEM media to each well, resuspend cells, ensuring no clumps are visible.</li>
	<li>Transfer all cells from each well into 1.5 mL centrifuge tubes, then centrifuge cells for 2 min at 1000 &#215; g at room temperature.</li>
	<li>Carefully remove the supernatant by pipetting and wash once with 1.0 mL PBS.</li>
	<li>Again, carefully remove the supernatant by pipetting, resuspend the cells in 500 &#181;L 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.</li>
	<li>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.</li>
	<li>Acquire and record at least 10,000 intact cells from each tube.</li>
</ol>
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.
<ol>
	<li>Drag all FACS files into the dashboard. Double-click on one of the files to display the FSC/SSC plot.</li>
	<li>Create a polygon gate to select intact cells, excluding debris in the lower-left corner. Name this subpopulation &#34;Intact Cells&#34; and drag this gate to the &#34;All Samples&#34; bar. Scroll through each sample using the Next Sample button to ensure appropriate gating for each sample (Figure 2A).</li>
	<li>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).</li>
	<li>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 &#34;Intact Cells&#34; 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 &#34;Intact Cells&#34; populations.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

FACS Analysis of GFP and mCherry Reporter Cells to Evaluate NHEJ & HR Efficiency

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

There are no conflicts of interest to disclose.

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 underly...

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&#39;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&#39;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.

<table><tbody><tr><td>6 cm dishes&amp;nbsp;</td><td>BBI</td><td>F611202-9001</td><td></td></tr><tr><td>6 well plates</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Ampicillin</td><td>Beyotime</td><td>ST007</td><td>Working concentration: 100 &amp;mu;g/mL</td></tr><tr><td>DH5&amp;alpha; Competent Cells</td><td>TIANGEN</td><td>CB101</td><td></td></tr><tr><td>DMEM</td><td>Hyclone</td><td>SH30022.01</td><td></td></tr><tr><td>DR-GFP</td><td>Addgene</td><td>26475</td><td></td></tr><tr><td>EJ5-GFP</td><td>Addgene</td><td>44026</td><td></td></tr><tr><td>EndoFree Maxi Plasmid&amp;nbsp; kit</td><td>TIANGEN</td><td>DP117</td><td>alternative endotoxin-free plasmid extraction kit can be used</td></tr><tr><td>FACS tubes</td><td>FALCON</td><td>352054</td><td></td></tr><tr><td>Fetal bovine serum</td><td>CLARK</td><td>FB25015</td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td>BD FACSCalibur</td><td></td></tr><tr><td>FlowJo V.10.1</td><td>Treestar</td><td></td><td>alternative analysis software can be used</td></tr><tr><td>HEK-293T cells</td><td>National Infrastructure of Cell Line Resource</td><td>1101HUM-PUMC000091</td><td></td></tr><tr><td>Lipo3000</td><td>Invitrogen</td><td>L3000015</td><td>alternative transfection regents can be used</td></tr><tr><td>PBS</td><td>Biosharp</td><td>BL601A</td><td></td></tr><tr><td>pCBASceI</td><td>Addgene</td><td>26477</td><td>I-SceI expressing plasmid</td></tr><tr><td>PCI2-HA-mCherry</td><td></td><td></td><td>alternative plasmids containing DsRed can be used</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td></td></tr></tbody></table>

analysis of nonhomologous end joining and homologous recombination efficiency in hek-293t cells using gfp-based reporter systems

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.

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...

Read this Scientific Article Protocol about Author Spotlight: Decoding DNA Repair by Extrachromosomal NHEJ Assay and HR Assays at JoVE.com

Author Spotlight: Decoding DNA Repair by Extrachromosomal NHEJ Assay and HR Assays

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.

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

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In ...

analysis-nonhomologous-end-joining-homologous-recombination

Research

JoVE Journal

Biology

1.9K Views.  Qiqihar Medical University. DNA-dubbelstrengsbreuken vertegenwoordigen de gevaarlijkste DNA-laesies. Als reactie hierop gebruiken cellen twee primaire mechanismen voor het herstel van DNA-dubbelstrengsbreuken, NHEJ en HR. Het kwantificeren van de efficiëntie van NHEJ en HR afzonderlijk is cruciaal voor het verkennen van de relevante mechanismen en factoren die ermee verband houden. De NHEJ-test en de HR-test zijn gevestigde methoden die worden gebruikt om de efficiëntie van NHEJ en HR te meten. Deze methoden zijn geba...

Video: Auteur in de schijnwerpers: DNA-reparatie decoderen door extrachromosomale NHEJ-test en HR-assays

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Genetics

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Biochemistry

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Quantifying DNA Double Strand Break Repair with Nanoluciferase Reporter Assays

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

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli.
Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence.
These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Author Spotlight: Developing Novel Anticancer Therapeutics Targeting the DNA Damage Response

We present protocols for the assessment of double strand break repair pathway proficiency in cells using a suite of luminescence-based extrachromosomal reporter substrates.

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is ...

Cancer Research

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

Immunology and Infection

CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we present a detailed protocol for the generation of novel in vitro models for HIV infection with chosen genomic integration sites using CRISPR-Cas9-based genome engineering technology. With this method, a reporter sequence of choice can be integrated into a targeted, chosen genomic locus, reflecting clinically relevant integration sites.
In the protocol, the design of an HIV-derived reporter and choosing of a target site and gRNA sequence are described. A targeting vector with homology arms is constructed and transfected into Jurkat T cells. The reporter sequence is targeted to the selected genomic site by homologous recombination facilitated by a Cas9-mediated double-strand break at the target site. Single-cell clones are generated and screened for targeting events by flow cytometry and PCR. Selected clones are then expanded, and correct targeting is verified by PCR, sequencing, and Southern blotting. Potential off-target events of CRISPR-Cas9-mediated genome engineering are analyzed.
By using this protocol, novel cell culture systems that model HIV infection at clinically relevant integration sites can be generated. Although the generation of single-cell clones and verification of correct reporter sequence integration is time-consuming, the resulting clonal lines are powerful tools to functionally analyze proviral integration site choice.

We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

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

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