Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep
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.
KeywordsGene-editing Livestock Biopsy Mosaic Ovine
We would like to thank Alma Islas-Trejo for library preparation and Elizabeth Tseng for assistance in bioinformatics. We also would like to acknowledge Kyle Wood for assistance with sheep care at UC Davis sheep Facility; and Devon Fitzpatrick, Ahmed Mahdi, Michelle Cruz, Charnice Robinson for helping during in vitro embryo production, embryo transferring and fetuses recovering. M.V was supported by a Fulbright-Uruguay Scholarship and an Austin Eugene Lyons Fellowship. Work was partially supported by USDA-NIFA-AFRI multistate project W3171 to P.J.R.
MV and FPS performed the experiments with additional input from STR, PJR and HN. MV, FPS, PJR, HL, JR, BRM, TM and MOH participated in sample processing and data analysis. MV, FS and PJR wrote the manuscript with suggestions from all the co-authors.
Compliance with ethical standards
Conflict of interest
Authors declare no competing financial interests statement.
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