Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep |
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Authors: | Marcela Vilarino Fabian Patrik Suchy Sheikh Tamir Rashid Helen Lindsay Juan Reyes Bret Roberts McNabb Talitha van der Meulen Mark O. Huising Hiromitsu Nakauchi Pablo Juan Ross |
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Affiliation: | 1.Department of Animal Science, College of Agricultural and Environmental Sciences,University of California Davis,Davis,USA;2.School of Medicine, Institute for Stem Cell Biology and Regenerative Medicine,Stanford University,Stanford,USA;3.Centre for Stem Cells and Regenerative Medicine and Institute for Liver Studies,King’s College,London,UK;4.Institute of Molecular Life Sciences,University of Zürich,Zurich,Switzerland;5.SIB Swiss Institute of Bioinformatics,University of Zürich,Zurich,Switzerland;6.Department of Population Health and Reproduction, School of Veterinary Medicine,University of California Davis,Davis,USA;7.Department of Neurobiology, Physiology and Behavior, College of Biological Sciences,University of California Davis,Davis,USA |
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Abstract: | The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in?~?50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals. |
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