IL2RG deficient X-SCID pigs generated via electroporation-mediated gene editing using CRISPR/Cas9

Area of Science:

• Genetics and Genomics
• Immunology
• Animal Models

Background:

• Interleukin 2 receptor gamma (IL2RG) knockout models are crucial for studying X-linked severe combined immunodeficiency (X-SCID).
• Pigs share anatomical and physiological similarities with humans, making them suitable for X-SCID modeling.
• Traditional methods for generating IL2RG knockout pigs involve somatic cell nuclear transfer.

Purpose of the Study:

• To investigate the feasibility of generating IL2RG knockout pigs via CRISPR/Cas9 zygotic gene editing and electroporation.
• To establish a novel method for creating X-SCID pig models.

Main Methods:

• CRISPR/Cas9 system with a selected guide RNA targeting the porcine IL2RG gene.
• Electroporation of gene-edited zygotes and transfer to surrogate mothers.
• Generation of F0 mosaic mutants and subsequent breeding to produce IL2RG knockout (XKOY) pigs.

Main Results:

• A mosaic mutant male piglet with a -19 bp frame-shift mutation in IL2RG was generated without off-target mutations.
• The mutation was successfully transmitted to F1 female offspring (XKOX).
• IL2RG-KO male pigs (XKOY) exhibited thymic hypoplasia and lacked key immune cells (T lymphocytes, NK cells).

Conclusions:

• Zygote electroporation is a feasible method for generating IL2RG-deficient pigs.
• The study successfully created IL2RG knockout pigs, offering a valuable model for X-SCID research.
• A fertile mosaic male pig capable of transmitting the mutation provides a potential alternative approach for generating X-linked immunodeficiency models.