Generation of Rhesus Macaque Embryos with Expanded CAG Trinucleotide Repeats in the Huntingtin Gene

Junghyun Ryu, John P. Statz, William Chan, Kiana Oyama, Maggie Custer, Martin Wienisch, Richard Chen, Carol B. Hanna, Jon D. Hennebold

Research output: Contribution to journalArticlepeer-review

Abstract

Huntington’s disease (HD) arises from expanded CAG repeats in exon 1 of the Huntingtin (HTT) gene. The resultant misfolded HTT protein accumulates within neuronal cells, negatively impacting their function and survival. Ultimately, HTT accumulation results in cell death, causing the development of HD. A nonhuman primate (NHP) HD model would provide important insight into disease development and the generation of novel therapies due to their genetic and physiological similarity to humans. For this purpose, we tested CRISPR/Cas9 and a single-stranded DNA (ssDNA) containing expanded CAG repeats in introducing an expanded CAG repeat into the HTT gene in rhesus macaque embryos. Analyses were conducted on arrested embryos and trophectoderm (TE) cells biopsied from blastocysts to assess the insertion of the ssDNA into the HTT gene. Genotyping results demonstrated that 15% of the embryos carried an expanded CAG repeat. The integration of an expanded CAG repeat region was successfully identified in five blastocysts, which were cryopreserved for NHP HD animal production. Some off-target events were observed in biopsies from the cryopreserved blastocysts. NHP embryos were successfully produced, which will help to establish an NHP HD model and, ultimately, may serve as a vital tool for better understanding HD’s pathology and developing novel treatments.

Original languageEnglish (US)
Article number829
JournalCells
Volume13
Issue number10
DOIs
StatePublished - May 2024

Keywords

  • CRISPR
  • Huntington’s disease
  • germline editing
  • rhesus macaque

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology

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