Nonhomologous End Joining
Nonhomologous End Joining - Related Videos
09:29
Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems
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Cited by 1 •
2024
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.
08:22
CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
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Cited by 15 •
2018
The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.
04:13
CRISPR Concatemer-Mediated Multiple Gene Knockout: A Technique to Simultaneously Knockout Multiple Genes by Non-Homologous End-Joining Pathway in Mouse Intestinal Cells
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2025
This video describes a gene knockout technique using a CRISPR-concatemer to simultaneously knock out multiple genes in cultured mouse intestinal organoid cells. This method is used to knock out a diseased gene and to elucidate the function of a gene and its paralogues.
06:10
Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
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2025
A detailed protocol is provided for using CRISPR/Cas9 technology to achieve highly efficient targeted knock-in of large, multicistronic constructs in primary human T cells via the homology-mediated end joining (HMEJ) DNA repair pathway. T cells engineered with this cGMP-adaptable protocol maintain excellent cell expansion, cytotoxicity, and cytokine production.
11:35
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Cited by 6 •
2017
Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.
FAQs
Ku70/Ku80 recognizes the exposed ends of a broken chromosome and protects them from inappropriate degradation. Its binding creates a platform for recruiting DNA-dependent protein kinase and other repair factors. By retaining the DNA ends at the break, this complex helps coordinate their alignment and subsequent preparation for ligation, making end recognition a critical entry point into the repair pathway.
DNA-dependent protein kinase participates in organizing and preparing the broken ends for repair, while XRCC4 and XLF associate with DNA ligase IV during the final joining step. Together, these factors connect end processing with ligation. Their coordinated action allows the pathway to restore chromosome continuity even when the two DNA ends require preparation before they can be joined.
The pathway joins DNA ends directly rather than preserving every original nucleotide at the break. End preparation and alignment can therefore remove bases or add a small number of nucleotides before ligation. These insertions or deletions are important consequences of repair because they can alter the local DNA sequence and provide a measurable record of how a break was resolved.
Because NHEJ operates throughout the cell cycle, cells can use it to respond to double-strand breaks without restricting repair to a single phase. This broad availability supports genome stability after DNA damage, including radiation-induced breaks. It also makes the pathway relevant in cellular conditions where rapid restoration of chromosome continuity is needed across different stages of cell growth.
A typical event begins when Ku70/Ku80 recognizes and protects the broken DNA ends. DNA-dependent protein kinase and associated repair factors then align and prepare those ends. The prepared ends are finally joined by DNA ligase IV together with XRCC4 and XLF. This sequence links damage recognition, end handling, and ligation into one coordinated repair process.
NHEJ contributes to immune-gene rearrangement, where its ability to reconnect broken DNA ends supports the restructuring of genetic material used in immune-system development. The same repair machinery also participates in broader cellular responses to DNA damage. These roles show that the pathway is not limited to chromosome maintenance, but also contributes to specialized biological changes.
Small insertions or deletions produced during end joining can reveal how a targeted DNA break was repaired. In genome editing, these sequence changes provide a way to alter or disrupt a selected region. In mutation analysis, the resulting patterns help investigators examine repair outcomes and connect sequence changes with the activity of the pathway.