CRISPR/Cas9 editing of the MYO7A gene in rhesus macaque embryos to generate a primate model of Usher Syndrome type 1B

Abstract

Mutations in the MYO7A gene lead to Usher syndrome type 1B (USH1B), a disease characterized by congenital deafness, vision loss, and balance impairment. To create a nonhuman primate (NHP) USH1B model, CRISPR/Cas9 was used to disrupt MYO7A in rhesus macaque zygotes. The targeting efficiency of Cas9 mRNA and hybridized crRNA-tracrRNA (hyb-gRNA) was compared to Cas9 nuclease (Nuc) protein and synthetic single guide (sg)RNAs. Nuc/sgRNA injection led to higher editing efficiencies relative to mRNA/hyb-gRNAs. Mutations were assessed by preimplantation genetic testing (PGT) and those with the desired mutations were transferred into surrogates. A pregnancy was established from an embryo where 92.1% of the PGT sequencing reads possessed a single G insertion that leads to a premature stop codon. Analysis of single peripheral blood leukocytes from the infant revealed that half the cells possessed the homozygous single base insertion and the remaining cells had the wild type MYO7A sequence. The infant showed sensitive auditory thresholds beginning at 3 months. Although further optimization is needed, our studies demonstrate that it is feasible to use CRISPR technologies for creating NHP models of human diseases.

A Comparison of Oocyte Yield between Ultrasound-Guided and Laparoscopic Oocyte Retrieval in Rhesus Macaques

“…Nonhuman primates (NHPs), such as Rhesus macaques (Macaca mulatta), are important animal models for biomedical research because of their evolutionary closeness to humans [1]. Modeling human disease with NHPs has thus become a major focus of research, propelled forward by advancements in genome editing and ARTs [2,3]. Obtaining high-quality oocytes is the first crucial step in this endeavor.…”

Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos

“…The emergence of the latest gene-editing technology, including the direct injection of CRISPR/ Cas9 into developing zygotes (Niu et al, 2014;Wan et al, 2015), has made the generation of genetically modified NHPs more feasible and accessible. However, recent publications indicated that it is difficult to obtain 100% editing efficiency, so the presence of desired mutations must be confirmed by a trophectoderm (TE) biopsy at the blastocyst stage (Vilarino et al, 2018;Ryu et al, 2022). In addition, the TE biopsy procedure is labor intensive and can adversely impact embryo development.…”