Optimizing Embryo Collection for Application of CRISPR/Cas9 System and Generation of Fukutin Knockout Rat Using This...

Area of Science:

• Development of genetically engineered animal models for disease research.
• CRISPR/Cas9 gene editing technology.
• Rat reproductive biology and embryology.

Background:

• Rat models offer advantages over mouse models in cognitive ability and physiological similarity to humans.
• Previous limitations in rat embryonic stem cell establishment and genetic modification hindered their use in disease modeling.
• Need for advanced genetic engineering techniques to overcome these limitations.

Purpose of the Study:

• To optimize conditions for rat in vitro fertilization and zygote development.
• To establish a rat model for muscular dystrophy using CRISPR/Cas9 gene editing.
• To overcome existing challenges in genetic modification of rats.

Main Methods:

• Optimized superovulation and fertilized-egg transfer protocols, including hormone concentrations (PMSG, hCG) and culture medium (mR1ECM).
• Selected single-guide RNA (sgRNA) with high targeting efficiency via PCR and T7E1 assay.
• Employed the CRISPR/Cas9 system to induce a deficiency in the rat *fukutin* gene, creating a muscular dystrophy model.

Main Results:

• Successfully established optimal conditions for obtaining high-quality rat zygotes and in vitro fertilization.
• Generated *fukutin* gene knockout rats using CRISPR/Cas9 with no detected off-target effects.
• Phenotypically confirmed the muscular dystrophy model by observing a significant reduction in body weight (one-third of control).

Conclusions:

• Successfully constructed the first CRISPR/Cas9-mediated rat model for muscular dystrophy.
• The optimized protocols enhance the potential for creating diverse genetically engineered rat disease models.
• This advancement is expected to significantly boost future research in various diseases using rat models.