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Related Concept Videos

CRISPR01:59

CRISPR

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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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Conservative Site-specific Recombination and Phase Variation02:53

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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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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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Updated: Jun 24, 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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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, United States.

Elife
|June 7, 2024
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 refolding procedure and targeted mutations significantly enhance PE efficiency in zebrafish embryos.

Keywords:
RNA structuregene editinggeneticsgenomicsprotein deliveryzebrafish

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biochemistry

Background:

  • CRISPR prime editing (PE) utilizes a Cas9 nickase-reverse transcriptase fusion protein (PE2) and a prime editing guide RNA (pegRNA).
  • pegRNAs are engineered to guide PE to target genomic sequences and encode desired edits.
  • Potential limitations in pegRNA structure can impede Cas9 complexation and reduce PE efficiency.

Purpose of the Study:

  • To investigate the impact of internal sequence complementarity within pegRNAs on PE efficiency.
  • To develop and validate methods for improving pegRNA structure and function.
  • To enhance the efficiency of CRISPR prime editing technology.

Main Methods:

  • Analysis of sequence complementarity within pegRNAs.
  • Development of a pegRNA refolding procedure.
  • Introduction of point mutations to disrupt internal pegRNA interactions.
  • Assessment of PE efficiency in zebrafish embryos using ribonucleoprotein complexes.

Main Results:

  • Sequence complementarity between the 5' and 3' regions of pegRNAs negatively affects Cas9 complexation.
  • A simple pegRNA refolding procedure increased PE efficiencies by up to 25-fold in zebrafish embryos.
  • Introducing point mutations to disrupt internal pegRNA interactions further improved PE efficiencies by up to sixfold.

Conclusions:

  • Internal sequence complementarity is a critical factor limiting pegRNA efficacy.
  • PegRNA refolding and targeted mutagenesis are effective strategies to enhance CRISPR prime editing efficiency.
  • These findings provide practical methods for optimizing PE for broader research and therapeutic applications.