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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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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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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.
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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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Related Experiment Video

Updated: Sep 4, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Modulating CRISPR-Cas Genome Editing Using Guide-Complementary DNA Oligonucleotides.

Thomas Swartjes1, Peng Shang2, Dennis T M van den Berg3

  • 1Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands; Delft, The Netherlands.

The CRISPR Journal
|July 20, 2022
PubMed
Summary
This summary is machine-generated.

Guide-complementary DNA oligonucleotides can inhibit Cas9 enzyme activity in human cells, enhancing genome editing safety. This study identifies key factors for effective inhibition, paving the way for controlled CRISPR-Cas applications.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The CRISPR-Cas system has transformed genome editing, offering potential for treating genetic disorders.
  • Ensuring the safety of CRISPR-Cas applications requires precise control over Cas enzyme activity.
  • Existing methods to modulate CRISPR-Cas activity include anti-CRISPR proteins and designed oligonucleotides.

Purpose of the Study:

  • To investigate the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes.
  • To assess the efficacy of DNA oligonucleotides in inhibiting Cas9 activity in human cells and understand the inhibition mechanism.
  • To identify factors crucial for robust inhibition by DNA oligonucleotides.

Main Methods:

  • Inhibition of Cas9 activity by DNA oligonucleotides was tested in human cells.
  • In vitro assays were performed to elucidate the mechanism of inhibition.
  • The impact of inhibitor length and the protospacer adjacent motif-loop on inhibition efficacy was evaluated.

Main Results:

  • DNA oligonucleotides were demonstrated to inhibit Cas9 activity in human cells.
  • Both on-target and off-target cleavage by Cas9 were reduced by the DNA oligonucleotides.
  • In vitro studies revealed that the length of the complementary region and the presence of a protospacer adjacent motif-loop are critical for effective inhibition.

Conclusions:

  • Guide-complementary DNA oligonucleotides serve as effective inhibitors of Cas9 activity.
  • These DNA oligonucleotides offer a controllable method for modulating CRISPR-Cas9 function.
  • The findings support the use of DNA oligonucleotides for controlled genome editing applications ex vivo and in vitro.