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Related Concept Videos

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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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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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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Related Experiment Video

Updated: May 21, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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Published on: November 1, 2024

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Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.

Honglue Shi, Noor Al-Sayyad, Kevin M Wasko

    Biorxiv : the Preprint Server for Biology
    |May 16, 2025
    PubMed
    Summary

    CRISPR-Cas9 genome editing efficiency decreases with reduced protospacer-adjacent motif (PAM) specificity. Broad PAM recognition leads to non-selective DNA binding and lower editing success in cells.

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    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genetics

    Background:

    • RNA-guided CRISPR-Cas enzymes are essential tools for programmable genome editing.
    • These enzymes recognize specific DNA sequences, including a protospacer-adjacent motif (PAM), to initiate editing.
    • Understanding the factors influencing editing efficiency is crucial for improving CRISPR technology.

    Purpose of the Study:

    • To investigate the molecular mechanisms underlying high-efficiency genome editing by CRISPR-Cas enzymes.
    • To determine the impact of protospacer-adjacent motif (PAM) binding specificity on the efficiency of *S. pyogenes* Cas9 (SpyCas9).
    • To elucidate the relationship between PAM recognition breadth and genome editing effectiveness.

    Main Methods:

    • Biochemical assays were used to assess enzyme activity and binding properties.
    • Biophysical techniques were employed to analyze protein-DNA and protein-RNA interactions.
    • Cell-based assays were conducted to evaluate genome editing efficiencies of *Spy*Cas9 variants.

    Main Results:

    • Reduced PAM specificity in *Spy*Cas9 variants resulted in persistent, non-selective DNA binding.
    • These variants showed recurrent failures in stable guide RNA hybridization to the target DNA.
    • Lower genome editing efficiencies were observed in cells expressing *Spy*Cas9 variants with reduced PAM specificity.

    Conclusions:

    • A fundamental trade-off exists between broad PAM recognition and genome editing effectiveness.
    • High-efficiency RNA-guided genome editing likely involves a two-step target capture: selective PAM binding followed by rapid DNA unwinding.
    • This study provides a foundational model for engineering improved CRISPR-Cas and related RNA-guided genome editors.