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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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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: Jul 15, 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

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PAM-flexible genome editing with an engineered chimeric Cas9.

Lin Zhao1, Sabrina R T Koseki1, Rachel A Silverstein2,3,4

  • 1Department of Biomedical Engineering, Duke University, Durham, NC, USA.

Nature Communications
|October 4, 2023
PubMed
Summary
This summary is machine-generated.

Researchers engineered a novel CRISPR enzyme, SpRYc, by combining SpRY and Sc+ Cas9 variants. This chimeric enzyme exhibits highly flexible protospacer adjacent motif (PAM) recognition, enabling precise genome editing across diverse sequences for potential therapeutic uses.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Gene Editing Technologies

Background:

  • CRISPR enzymes necessitate a specific protospacer adjacent motif (PAM) for target site recognition.
  • This PAM requirement restricts the accessibility of certain DNA sequences for genome editing applications.
  • Existing Cas9 variants have limitations in PAM flexibility, efficiency, or accuracy.

Purpose of the Study:

  • To engineer a chimeric CRISPR enzyme with enhanced and flexible protospacer adjacent motif (PAM) recognition.
  • To overcome the limitations of sequence accessibility imposed by traditional CRISPR-Cas9 systems.
  • To develop a versatile genome editing tool for diverse therapeutic applications.

Main Methods:

  • Recombination of the PAM-interacting domain of SpRY (NRN > NYN PAM preference) with the N-terminus of Sc++ (NNG editing capabilities).
  • Generation of a chimeric enzyme, designated SpRYc, integrating properties of both parent Cas9 variants.
  • Demonstration of SpRYc's ability to edit diverse PAMs and disease-related genetic loci.

Main Results:

  • The chimeric SpRYc enzyme exhibits highly flexible and broad PAM recognition capabilities.
  • SpRYc successfully and specifically edits diverse protospacer adjacent motifs (PAMs).
  • The enzyme demonstrates editing of disease-related loci, indicating potential therapeutic relevance.

Conclusions:

  • Integrative protein design is a powerful strategy for engineering advanced Cas9 variants.
  • SpRYc offers robust flexibility in PAM recognition, expanding the scope of CRISPR genome editing.
  • The developed enzyme motivates downstream applications requiring precise genomic positioning and editing of previously inaccessible sites.