Earticle

초록

영어

Direct injection of CRISPR/Cas9 into zygotes enables the production of genetically modified nonhuman primates (NHPs) essential for modeling specific human diseases, such as Usher syndrome, and for developing novel therapeutic strategies. Usher syndrome is a rare genetic disease that causes loss of hearing, retinal degeneration, and problems with balance, and is attributed to a mutation in MYO7A, a gene that encodes an uncommon myosin motor protein expressed in the inner ear and retinal photoreceptors. To produce an Usher syndrome type 1B (USH1B) rhesus macaque model, we disrupted the MYO7A gene in developing zygotes. Identification of appropriately edited MYO7A embryos for knockout embryo transfer requires sequence analysis of material recovered from a trophectoderm (TE) cell biopsy. However, the TE biopsy procedure is labor intensive and could adversely impact embryo development. Recent studies have reported using cell-free DNA (cfDNA) from embryo culture media to detect aneuploid embryos in human in vitro fertilization (IVF) clinics. The cfDNA is released from the embryo during cell division or cell death, suggesting that cfDNA may be a viable resource for sequence analysis. Moreover, cfDNA collection is not invasive to the embryo and does not require special tools or expertise. We hypothesized that selection of appropriate edited embryos could be performed by analyzing cfDNA for MYO7A editing in embryo culture medium, and that this method would be advantageous for the subsequent generation of genetically modified NHPs. The purpose of this experiment is to determine whether cfDNA can be used to identify the target gene mutation of CRISPR/Cas9 injected embryos. In this study, we were able to obtain and utilize cfDNA to confirm the mutagenesis of MYO7A, but the method will require further optimization to obtain better accuracy before it can replace the TE biopsy approach.

목차

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Ethical approval
Design of sgRNA and injection materials preparation
Direct injection of CRISPR/Cas9 into developing zygotes
Genotyping from TE biopsy and cfDNA
RESULTS
DISCUSSION
REFERENCES

키워드

저자

• Junghyun Ryu [ Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USA ] Corresponding Author
• Fernanda C. Burch [ Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USA ]
• Emily Mishler [ Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USA ]
• Martha Neuringer [ Division of Neuroscience, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USA ]
• Jon D. Hennebold [ Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USA, Department of Obstetrics & Gynecology, Oregon Health & Science University, Portland 97239, USA ]
• Carol Hanna [ Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USA ] Corresponding Author

참고문헌

발행기관

간행물

관심저널 등록

• 간행물명

  Journal of Animal Reproduction and Biotechnology

• 간기

  계간

• pISSN

  2671-4639

• eISSN

  2671-4663

• 수록기간

  2019~2026

• 십진분류

  KDC 527 DDC 636