Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 technology revolutionized genome editing.
• Low efficiency of homology directed repair (HDR) in mouse zygotes limits its application.
• Optimization of HDR parameters is needed for efficient gene targeting.

Purpose of the Study:

• To systematically explore and optimize parameters for enhancing homology directed repair (HDR) efficiency in mouse zygotes.
• To compare the targeting efficiency of different Cas9 variants (Cas9, Cas9nickase, dCas9-FokI).
• To develop a streamlined method for multiplexed gene targeting in zygotes.

Main Methods:

• Systematic characterization of parameters including single guide RNA activity and homology arm design.
• Comparison of Cas9, Cas9nickase, and dCas9-FokI targeting efficiencies.
• Application of optimized conditions to mouse zygotes using Cas9 mRNA or protein delivery.

Main Results:

• Cas9 nucleo-protein complex significantly enhances multiplexed targeting efficiency in mouse zygotes.
• Optimized conditions enable one-step zygote injection for generating conditional alleles via homologous recombination.
• Simultaneous gene knockout via non-homologous end joining is achieved in non-targeted alleles.

Conclusions:

• The developed method provides a highly efficient and streamlined approach for CRISPR/Cas9-mediated genome editing in mouse zygotes.
• This technique facilitates the generation of complex genetic modifications, including conditional alleles and simultaneous knockouts.
• The findings advance the application of genome editing for creating genetically modified mouse models.