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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

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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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CRISPR and crRNAs02:53

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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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Related Experiment Video

Updated: Nov 23, 2025

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

Published on: May 25, 2018

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Precise and broad scope genome editing based on high-specificity Cas9 nickases.

Qian Wang1, Jin Liu1, Josephine M Janssen1

  • 1Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.

Nucleic Acids Research
|January 5, 2021
PubMed
Summary

High-specificity CRISPR-Cas9 nickases enhance genome editing precision by reducing off-target effects. These advanced RNA-guided nucleases improve DNA knockouts and knock-ins, ensuring greater safety and predictability in genetic manipulations.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas systems, including RNA-guided nucleases (RGNs), enable genome editing but face challenges with off-target activity and multi-copy sequences.
  • Existing dual nicking RGNs and high-specificity RGNs show reduced off-target effects.

Purpose of the Study:

  • To convert high-specificity Cas9 nucleases into nicking Cas9D10A variants with superior precision.
  • To evaluate the efficacy of dual nicking RGNs derived from these variants for gene editing.
  • To assess the ability of these systems to distinguish similar DNA sequences and maintain genomic integrity.

Main Methods:

  • Conversion of high-specificity Cas9 into Cas9D10A nickase variants.
  • Application of dual nicking RGNs for gene knockouts and knock-ins.
  • Assessment of sequence specificity, including discrimination of single-nucleotide polymorphisms.
  • Genome-wide high-throughput sequencing for assessing genomic integrity.

Main Results:

  • High-specificity Cas9 nucleases were successfully converted into nicking variants with enhanced precision.
  • Dual nicking RGNs achieved gene knockout and knock-in efficiencies comparable to or exceeding conventional methods.
  • These high-specificity RGNs demonstrated the ability to differentiate highly similar sequences by tolerating single-base polymorphisms.
  • Unbiased genome-wide assays confirmed the preservation of genomic integrity.

Conclusions:

  • The developed high-specificity Cas9 nickase variants expand the toolkit for precise genome editing.
  • These tools enhance the range and accuracy of DNA knockout and knock-in procedures.
  • The high-specificity genome editing strategies offer improved predictability and safety for genetic manipulations.