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- Title
Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.
- Authors
Vilarino, Marcela; Suchy, Fabian Patrik; Rashid, Sheikh Tamir; Lindsay, Helen; Reyes, Juan; McNabb, Bret Roberts; van der Meulen, Talitha; Huising, Mark O.; Nakauchi, Hiromitsu; Ross, Pablo Juan
- 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.
- Subjects
MOSAICISM; EMBRYOLOGY; GENETIC mutation; CRISPRS; SHEEP
- Publication
Transgenic Research, 2018, Vol 27, Issue 6, p525
- ISSN
0962-8819
- Publication type
Article
- DOI
10.1007/s11248-018-0094-x