Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Disease Modeling

Background:

• CRISPR/Cas9 technology revolutionized genome manipulation.
• Cas9-transgenic animals offer potential for in vivo genome editing but face limitations in platform availability and disease modeling.
• Efficient and conditional genome editing tools are crucial for advancing in vivo research.

Purpose of the Study:

• To develop and validate novel doxycycline-inducible Cas9 platforms for conditional genome editing.
• To facilitate efficient multi-locus editing in vitro and in vivo.
• To establish a robust system for complex disease modeling in mice.

Main Methods:

• Designed two doxycycline (Dox)-inducible Cas9 platforms.
• Utilized a site-specific multi-segment cloning strategy for integrating multiple single guide RNAs (sgRNAs).
• Employed rtTA and TRE-Cas9 systems at specific mouse loci (Rosa26 and Col1a1) for inducible expression.

Main Results:

• Achieved efficient conditional genome editing at multiple loci.
• Demonstrated high efficiency of inducible insertions and deletions (indels) with minimal leaky editing using a specific platform configuration.
• Successfully modeled Wilms' tumor and intestinal adenoma progression with multiple mutations, including large genomic deletions.

Conclusions:

• The developed Cas9 platforms enable efficient and conditional multi-locus genome editing in vitro and in vivo.
• The established system simplifies complex disease modeling in mice, particularly for cancer research.
• These platforms enhance the utility of in vivo experiments across various biological disciplines.