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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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Updated: Jul 17, 2025

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.
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Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions.

Weiting Zhang1,2, Karl Petri3,4, Junyan Ma1,2,5

  • 1Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA.

Biorxiv : the Preprint Server for Biology
|August 30, 2023
PubMed
Summary
This summary is machine-generated.

Internal sequence complementarity in prime editing guide RNAs (pegRNAs) can reduce CRISPR prime editing (PE) efficiency. A simple pegRNA refolding procedure significantly enhances PE efficiency in zebrafish embryos.

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies

Background:

  • CRISPR prime editing (PE) utilizes a Cas9 nickase-reverse transcriptase fusion protein (PE2) and a prime editing guide RNA (pegRNA).
  • pegRNAs are extended guide RNAs encoding target specificity and desired genetic edits.
  • Internal sequence complementarity within pegRNAs can hinder Cas9 complexation and reduce PE efficiency.

Approach:

  • Investigated the impact of 5' and 3' region sequence complementarity in pegRNAs on Cas9 complexation.
  • Developed and tested a pegRNA refolding procedure to mitigate internal sequence interactions.
  • Introduced point mutations in pegRNAs to disrupt intramolecular interactions.

Key Points:

  • Sequence complementarity between the 5' and 3' regions of pegRNAs negatively affects Cas9 binding.
  • A simple pegRNA refolding method improved ribonucleoprotein-mediated PE efficiencies up to 25-fold in zebrafish embryos.
  • Disrupting internal pegRNA interactions via point mutations further enhanced PE efficiencies by up to 6-fold.

Conclusions:

  • Internal sequence complementarity is a critical factor limiting pegRNA function.
  • PegRNA refolding and targeted mutation strategies offer simple yet effective methods to enhance CRISPR prime editing efficiency.
  • These findings provide practical approaches to optimize PE for broader applications in genome engineering.