Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system

Area of Science:

• * Molecular Biology
• * Genetics
• * Gene Editing

Background:

• * Point mutations are crucial for understanding gene and protein function, but in vivo validation is challenging.
• * Existing in vivo methods for genomic modification are technically demanding and time-consuming.
• * Many in vitro findings remain unconfirmed in a living organism.

Purpose of the Study:

• * To establish a rapid and cost-effective CRISPR/Cas9 system for generating point mutations in mouse genomes.
• * To create mouse models with single amino acid substitutions for in vivo functional studies.
• * To validate in vitro findings and study patient-derived mutations in a relevant in vivo context.

Main Methods:

• * Utilized CRISPR/Cas9 system with guide RNA (gRNA), hCas9 mRNA, and single-stranded donor oligonucleotides (ssODN).
• * Microinjected components into mouse zygotes to achieve targeted genomic modifications.
• * Employed both single gRNA/WT hCas9 and double nicking strategies for mutation induction.

Main Results:

• * Successfully generated mice with precise point mutations, resulting in single amino acid substitutions.
• * Demonstrated the efficiency of both single and double nicking CRISPR/Cas9 approaches.
• * Identified the distance between the modification and gRNA target sites as a key factor influencing substitution efficiency.

Conclusions:

• * The developed CRISPR/Cas9 method provides a powerful and efficient tool for in vivo genome editing.
• * This technique facilitates the validation of in vitro experimental results within a biological system.
• * Enables the study of genetic disorder mutations in a relevant in vivo setting.