One-Step In Vitro Generation of ETV2-Null Pig Embryos.

Area of Science:

• Regenerative Medicine
• Genetics
• Developmental Biology

Background:

• Organ transplantation faces a critical shortage of donor organs, leading to thousands of deaths annually.
• Regenerative medicine aims to generate humanized organs in pigs using pluripotent stem cells (PSCs) and blastocyst complementation.
• Endothelial cells (ECs) are key targets in xenotransplantation rejection, necessitating the development of organ-disabled models.

Purpose of the Study:

• To produce hematoendothelial-disabled pig embryos by targeting the ETV2 gene.
• To establish an efficient method for generating genetically modified pig embryos for xenotransplantation research.

Main Methods:

• CRISPR-Cas9 genome editing was used to target the ETV2 gene in porcine fibroblasts and embryos.
• Five guide RNAs (gRNAs) were designed and tested for cleavage efficiency against the porcine ETV2 gene.
• Ribonucleoprotein complexes (RNPs) containing optimized gRNAs were microinjected into one-cell-stage porcine embryos.

Main Results:

• Four out of five designed gRNAs demonstrated cleavage capacity in vitro.
• Microinjection of RNPs into porcine embryos resulted in successful gene editing.
• Combining two highly efficient gRNAs and increasing microinjection concentration significantly improved biallelic mutation rates.

Conclusions:

• An efficient one-step method for generating hematoendothelial-disabled pig embryos was established using CRISPR-Cas9.
• This genetically modified pig model is a valuable tool for research into the in vivo generation of humanized organs.
• The developed model holds potential for advancing xenotransplantation and addressing the organ shortage crisis.