Targeted Transgenic Mice Using CRISPR /Cas9 Technology.

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biotechnology

Background:

• CRISPR/Cas9 technology offers a faster, more cost-effective alternative to traditional gene targeting methods for mammalian genome editing.
• Developing precise gene editing techniques is crucial for creating accurate animal models for research.

Purpose of the Study:

• To provide a comprehensive, step-by-step protocol for generating gene-edited mouse models using CRISPR/Cas9.
• To detail the design of single guide RNAs (sgRNA) for knockout (KO) and single-strand oligonucleotide (ssODN) matrices for knock-in (KI) strategies.
• To describe two distinct CRISPR/Cas9 delivery methods for zygote-stage embryos.

Main Methods:

• CRISPR/Cas9 system utilization for gene editing in mouse embryos.
• Design and synthesis of single guide RNAs (sgRNA) for targeted gene disruption.
• Design and synthesis of single-strand oligonucleotide (ssODN) matrices for precise gene insertion.
• Delivery of CRISPR/Cas9 components via cytoplasmic injection into zygotes.
• Delivery of CRISPR/Cas9 components via electroporation of zygotes.

Main Results:

• Successful generation of protocols for both CRISPR/Cas9-mediated knockout (KO) and knock-in (KI) mouse models.
• Detailed instructions provided for sgRNA design for KO and ssODN matrix design for KI.
• Demonstration of two viable CRISPR/Cas9 delivery methods (cytoplasmic injection and electroporation) for producing gene-edited mice.

Conclusions:

• CRISPR/Cas9 technology provides a robust and efficient platform for generating custom mouse models.
• The described protocols enable researchers to create KO and KI mice with enhanced speed and cost-effectiveness.
• This chapter serves as a practical guide for researchers aiming to utilize CRISPR/Cas9 for mammalian gene editing.