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Related Concept Videos

Homologous Recombination02:31

Homologous Recombination

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Fixing Double-strand Breaks02:04

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The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
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Base Excision Repair01:54

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One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
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Long-patch Base Excision Repair01:02

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Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:
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Overview
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Gene Conversion02:08

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Other than maintaining genome stability via DNA repair, homologous recombination plays an important role in diversifying the genome. In fact, the recombination of sequences forms the molecular basis of genomic evolution. Random and non-random permutations of genomic sequences create a library of new amalgamated sequences. These newly formed genomes can determine the fitness and survival of cells. In bacteria, homologous and non-homologous types of recombination lead to the evolution of new...
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Related Experiment Video

Updated: Aug 29, 2025

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
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Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors.

Sukanya Iyer1, Aamir Mir2, Joel Vega-Badillo2

  • 1Department of Molecular, Cell and Cancer Biology; Worcester, Massachusetts, USA.

The CRISPR Journal
|September 7, 2022
PubMed
Summary
This summary is machine-generated.

Circular single-stranded DNA (cssDNA) donors, produced using ssDNA phage, significantly improve homology-directed repair (HDR) efficiency for CRISPR genome editing. These cssDNA donors outperform linear ssDNA, offering a cost-effective method for precise gene knockins in mammalian cells.

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

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-based nucleases have advanced genome editing, but homology-directed repair (HDR) efficiency remains a challenge.
  • Traditional DNA donors like plasmids have low HDR efficiency and off-target integration issues.
  • Single-stranded DNA (ssDNA) donors show promise for efficient HDR with reduced off-target effects.

Purpose of the Study:

  • To develop and evaluate circular ssDNA (cssDNA) donors produced via ssDNA phage for enhanced HDR efficiency.
  • To compare the efficacy of cssDNA donors against linear ssDNA (lssDNA) donors with different CRISPR nucleases (Cas9, Cas12a).
  • To assess precise gene editing and fluorescent tag knockin efficiencies in mammalian cell lines.

Main Methods:

  • Production of long cssDNA donors using ssDNA phage technology.
  • Development of a modified traffic light reporter (TLR-multi-Cas variant 1 [MCV1]) system for comparing nuclease and donor efficiencies.
  • Evaluation of HDR efficiencies with cssDNA, lssDNA, and plasmid donors using Cas9 and Cas12a nucleases.
  • Assessment of fluorescent tag knockin at endogenous loci in HEK293T and K562 cells.

Main Results:

  • cssDNA donors demonstrated superior HDR integration frequencies compared to lssDNA donors.
  • The TLR-MCV1 system enabled efficient side-by-side comparison of different nuclease and donor combinations.
  • High targeting efficiency was achieved with cssDNA donors, enabling biallelic integrants.
  • cssDNA donors facilitated robust and efficient insertion of reporter tags at endogenous sites.

Conclusions:

  • cssDNA donors produced via ssDNA phage are highly efficient and cost-effective for homology-directed repair.
  • Circular ssDNA donors represent an improved alternative to linear ssDNA and plasmid donors for gene knockins.
  • This method offers a robust strategy for precise genome editing applications in mammalian cell lines.