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Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

Alexa Burger1, Helen Lindsay2, Anastasia Felker1

  • 1Institute of Molecular Life Sciences, University of Zürich, Zürich 8057, Switzerland.

Development (Cambridge, England)
|May 1, 2016
PubMed
Summary

Optimally solubilized CRISPR-Cas9 ribonucleoprotein complexes (RNPs) achieve maximal mutagenesis efficiency in zebrafish embryos. This breakthrough enables scalable loss-of-function studies and precise gene editing applications.

Keywords:
CRISPR-Cas9CrispRVariantsCrispantCalGenome editingMutagenesisRNPZebrafish

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Area of Science:

  • Molecular Biology
  • Genetics
  • Developmental Biology

Background:

  • CRISPR-Cas9 technology allows for targeted DNA modifications.
  • In vivo delivery and efficiency of CRISPR-Cas9 ribonucleoprotein complexes (RNPs) face challenges like toxicity and mosaicism.
  • Optimizing RNP formulation is crucial for efficient mutagenesis in model organisms.

Purpose of the Study:

  • To enhance the efficiency and reproducibility of CRISPR-Cas9 mediated mutagenesis in zebrafish embryos.
  • To develop a method for achieving near-saturating mutagenesis for loss-of-function studies.
  • To explore the application of saturating mutagenesis in identifying functional non-coding regulatory elements.

Main Methods:

  • In vitro assembly of fluorescent Cas9-sgRNA RNPs in a solubilizing salt solution.
  • Delivery of RNPs into zebrafish embryos.
  • High-throughput sequencing (MiSeq) of targeted loci in individual embryos.
  • Utilizing customized software (CrispRVariants) for mutagenesis quantification and visualization.

Main Results:

  • Maximal mutagenesis efficiency was achieved in zebrafish embryos using the optimized RNP formulation.
  • Bi-allelic mutagenesis reached saturation at multiple gene loci, enabling robust loss-of-function analysis.
  • Saturating mutagenesis successfully identified functional elements in both transgenic reporters and endogenous genes.
  • The method demonstrated high reproducibility and minimal toxicity.

Conclusions:

  • Optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs are a powerful and reproducible tool for maximal DNA cutting efficiency in vivo.
  • This approach facilitates direct and scalable loss-of-function studies in zebrafish and other model systems.
  • The methodology extends applications beyond simple gene knockout to functional characterization of regulatory elements.