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 molecule

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.......

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