CRISPR/Cas9-editing of PRNP in Alpine goats

Area of Science:

• Veterinary Genetics
• Neurodegenerative Disease Research
• Gene Editing Technologies

Background:

• Prion diseases, such as those affecting goats, stem from misfolded cellular prion protein (PrP^c).
• Complete absence of PrP^c confers resistance to prion diseases.
• Targeting the PRNP gene offers a strategy to prevent prion diseases.

Purpose of the Study:

• To engineer Alpine goats with knockout (KO) alleles of the PRNP gene using CRISPR/Cas9.
• To establish a PRNP-KO Alpine goat line for future prion disease resistance studies.
• To investigate novel PRNP variants for their potential role in prion disease.

Main Methods:

• CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides were used for gene editing.
• Genome editors were injected into Alpine goat embryos/oocytes before electroporation.
• Edited embryos were transferred to recipient goats, and offspring were analyzed for PRNP gene modifications.

Main Results:

• Eight founder Alpine goats with PRNP genome-edited alleles were successfully generated from 10 offspring.
• Five different KO mutations and a novel PRNP^del6 variant were identified among the founders.
• Three founders with only edited alleles were phenotypically normal, with one male PRNP-KO animal selected for further breeding.

Conclusions:

• CRISPR/Cas9 technology can efficiently generate PRNP-knockout Alpine goats.
• The established PRNP-KO line provides a valuable model for studying prion disease resistance.
• Further research will explore the PRNP^del6 variant and the influence of genetic background on PRNP KO phenotypes.