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Optimizing Prime Editing in Zebrafish.

Rehman Basharat1, Gina Rizzo1,2, Josiah D Zoodsma1,2

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Prime editing efficiently introduces missense mutations in zebrafish using novel variants. PEmax demonstrated the highest efficiency, establishing it as a robust tool for creating genetic disease models.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Zebrafish Models

Background:

  • Prime editing is a CRISPR-based technology for precise DNA modifications.
  • Zebrafish are valuable models for genetic disease research.
  • The efficiency of novel prime editor variants in zebrafish is not well-established.

Purpose of the Study:

  • To systematically evaluate the efficiency of five prime editor (PE) variants in zebrafish.
  • To compare the performance of PE2, PE6b, PE6c, PEmax, and PE7 for introducing missense mutations.
  • To assess strategies for enhancing prime editing guide RNA (pegRNA) stability.

Main Methods:

  • Testing five PE variants (PE2, PE6b, PE6c, PEmax, PE7) using mRNA delivery in zebrafish.
  • Designing pegRNAs to introduce five distinct missense mutations.
  • Evaluating editing efficiency at targeted genomic sites.

Main Results:

  • Efficient prime editing was achieved at four out of five targeted sites across multiple PE variants.
  • PEmax exhibited the highest efficiency for introducing pure prime edits, reaching up to 15.34%.
  • Strategies like epegRNAs and La RNA binding motif addition did not enhance editing performance.

Conclusions:

  • PEmax is identified as a highly efficient prime editor for introducing missense mutations in zebrafish.
  • This study provides valuable data for selecting prime editor variants for zebrafish genetic engineering.
  • PEmax offers a robust tool for generating zebrafish disease models with precise genetic alterations.