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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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CRISPR01:59

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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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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DNA Topoisomerases02:02

DNA Topoisomerases

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Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA.
Types and Mechanism of action
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CRISPR and crRNAs02:53

CRISPR and crRNAs

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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Related Experiment Video

Updated: Jul 14, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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Negative DNA supercoiling induces genome-wide Cas9 off-target activity.

Matthew D Newton1, Marialucrezia Losito2, Quentin M Smith3

  • 1Department of Infectious Disease, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0HS, UK; Single Molecule Imaging, MRC-London Institute of Medical Sciences, Du Cane Road, London W12 0HS, UK; DSB Repair Metabolism Laboratory, The Francis Crick Institute, London NW1 1AT, UK.

Molecular Cell
|October 6, 2023
PubMed
Summary

DNA topology, like negative supercoiling, significantly increases CRISPR-Cas9 gene editing off-target activity. This discovery reveals how cellular processes may inadvertently create unintended DNA breaks, impacting therapeutic safety.

Keywords:
CRISPR/Cas9Cell editingDNA topologyGene editingGenome-wide off-targetNegative supercoilingNext Generation SequencingOptical-tweezersSingle-molecule microscopyTranscription-coupled off-targets

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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas9 gene editing offers therapeutic potential but faces challenges with off-target activity.
  • Previous research indicated DNA stretching induces off-target effects.
  • Hypothesis: DNA topological distortions, such as negative supercoiling, may reduce Cas9 specificity.

Purpose of the Study:

  • To investigate the impact of DNA topology, specifically negative supercoiling, on CRISPR-Cas9 specificity.
  • To determine if DNA distortions in vitro and in vivo influence Cas9 off-target binding and cleavage.

Main Methods:

  • Single-molecule optical tweezers used to study DNA supercoiling effects on Cas9 binding.
  • Adapted CIRCLE-seq employed to detect genome-wide off-target double-strand breaks.
  • In vivo experiments assessed the influence of directed local DNA distortion on cellular off-target activity.

Main Results:

  • Negative supercoiling of DNA induced sequence-specific Cas9 off-target binding at multiple sites, even at low forces.
  • Over 10,000 Cas9 off-target double-strand breaks were detected genome-wide, attributed to increased mismatch tolerance under supercoiling.
  • Directed local DNA distortion in cells increased CRISPR-Cas9 off-target activity, with events detectable during genome editing.

Conclusions:

  • CRISPR-Cas9 off-target activity is significantly regulated by DNA topology both in vitro and in vivo.
  • Cellular processes involving DNA topology changes, such as transcription and replication, may lead to off-target activity at novel sites.
  • Understanding DNA topology's role is crucial for enhancing CRISPR-Cas9 specificity and therapeutic safety.