Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos
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 h...
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| Format: | Article |
| Language: | English |
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The Korean Society of Animal Reproduction and Biotechnology
2022-12-01
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| Series: | Journal of Animal Reproduction and Biotechnology |
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| Online Access: | https://www.e-jarb.org/journal/view.html?uid=2639&vmd=Full |
| _version_ | 1797974414552203264 |
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| author | Junghyun Ryu Fernanda C. Burch Emily Mishler Martha Neuringer Jon D. Hennebold Carol Hanna |
| author_facet | Junghyun Ryu Fernanda C. Burch Emily Mishler Martha Neuringer Jon D. Hennebold Carol Hanna |
| author_sort | Junghyun Ryu |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T04:18:30Z |
| format | Article |
| id | doaj.art-739593c2ee5e44c6b5c654374dce16b2 |
| institution | Directory Open Access Journal |
| issn | 2671-4639 2671-4663 |
| language | English |
| last_indexed | 2024-04-11T04:18:30Z |
| publishDate | 2022-12-01 |
| publisher | The Korean Society of Animal Reproduction and Biotechnology |
| record_format | Article |
| series | Journal of Animal Reproduction and Biotechnology |
| spelling | doaj.art-739593c2ee5e44c6b5c654374dce16b22022-12-31T04:22:10ZengThe Korean Society of Animal Reproduction and BiotechnologyJournal of Animal Reproduction and Biotechnology2671-46392671-46632022-12-0137429229710.12750/JARB.37.4.292Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryosJunghyun Ryu0https://orcid.org/0000-0001-8064-5762Fernanda C. Burch1https://orcid.org/0000-0002-9173-1663Emily Mishler2https://orcid.org/0000-0002-0735-0180Martha Neuringer3https://orcid.org/0000-0001-5548-7774Jon D. Hennebold4https://orcid.org/0000-0002-6760-3733Carol Hanna5https://orcid.org/0000-0002-2790-3555Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USADivision of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USADivision of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USADivision of Neuroscience, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USADivision of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USADivision of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton 97006, USADirect 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.https://www.e-jarb.org/journal/view.html?uid=2639&vmd=Fullcell-free dnacrispr/cas9knockoutmyo7ausher syndrome |
| spellingShingle | Junghyun Ryu Fernanda C. Burch Emily Mishler Martha Neuringer Jon D. Hennebold Carol Hanna Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos Journal of Animal Reproduction and Biotechnology cell-free dna crispr/cas9 knockout myo7a usher syndrome |
| title | Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos |
| title_full | Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos |
| title_fullStr | Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos |
| title_full_unstemmed | Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos |
| title_short | Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos |
| title_sort | utilizing cell free dna to validate targeted disruption of myo7a in rhesus macaque pre implantation embryos |
| topic | cell-free dna crispr/cas9 knockout myo7a usher syndrome |
| url | https://www.e-jarb.org/journal/view.html?uid=2639&vmd=Full |
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