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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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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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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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Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. This gene encodes the ced-3 caspase that is similar to the interleukin-1-beta converting enzyme or ICE in mammals. In addition to apoptosis, caspases also function in the inflammatory response. Inflammatory caspases are essential in activating pro-inflammatory cytokines that recruit immune cells and block the replication of pathogens inside...
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Related Experiment Video

Updated: Aug 23, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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CIRCLE-Seq for Interrogation of Off-Target Gene Editing

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Structural basis for Cas9 off-target activity.

Martin Pacesa1, Chun-Han Lin2, Antoine Cléry3

  • 1Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

Cell
|October 28, 2022
PubMed
Summary

CRISPR-Cas9 genome editing can target unintended DNA sequences, raising safety concerns. Structural analysis reveals noncanonical base pairing and accommodation of deletions enable off-target binding, guiding improved guide RNA design.

Keywords:
CRISPRCas9X-ray crystallographybase pairinggenome editingguide RNAmismatchnucleaseoff-target

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • CRISPR-associated (Cas) nuclease Cas9 is a powerful genome editing tool.
  • Cas9 specificity relies on guide RNA complementarity to target DNA.
  • Off-target cleavage by Cas9 poses safety risks for clinical applications.

Purpose of the Study:

  • To elucidate the structural basis of Cas9 off-target binding and cleavage.
  • To understand how Cas9 accommodates mismatches and deletions in target DNA.
  • To inform the rational design of safer Cas9-based genome editing systems.

Main Methods:

  • X-ray crystallography of Cas9-DNA complexes.
  • Analysis of off-target substrates with varying complementarity.
  • Structural comparison of on-target and off-target binding modes.

Main Results:

  • Cas9 binds off-target DNA via noncanonical base-pairing interactions.
  • Single-nucleotide deletions are accommodated by base skipping or multiple noncanonical pairs.
  • PAM-distal mismatches induce duplex unpairing and conformational changes in Cas9.

Conclusions:

  • Structural insights explain Cas9 off-target activity.
  • Findings facilitate improved guide RNA design for enhanced specificity.
  • This work aids in developing better off-target prediction algorithms for CRISPR technology.