Generating an organ-deficient animal model using a multi-targeted CRISPR-Cas9 system

Area of Science:

• Developmental Biology
• Genetics
• Biotechnology

Background:

• Gene-knockout animal models for blastocyst complementation often lack viability.
• Homozygous mutant embryos are scarce, limiting research potential.

Purpose of the Study:

• To develop a robust method for generating organ-deficient embryos using CRISPR-Cas9.
• To create viable interspecies chimeras for organ development studies.

Main Methods:

• Utilized the CRISPR-Cas9-sgRNAms system to induce targeted cell death in mouse embryos.
• Developed a mouse model (Foxn1Cas9; Rosa26_ms) with Cas9 under the Foxn1 promoter.
• Employed blastocyst complementation with rat embryonic stem cells.

Main Results:

• The CRISPR-Cas9-sgRNAms system effectively induced cell ablation in vitro.
• Generated mouse embryos with an athymic phenotype.
• Successfully created an interspecies chimera with a rat cell-derived thymus.

Conclusions:

• The CRISPR-Cas9-sgRNAms system provides a viable strategy for generating organ-deficient embryos.
• This method overcomes limitations of traditional gene-knockout models for blastocyst complementation.
• Enables the development of functional organs from different species within a chimera.