Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep

Marcela Vilarino, Fabian Patrik Suchy, Sheikh Tamir Rashid, Helen Lindsay, Juan Reyes, Bret McNabb, Talitha van der Meulen, Mark O. Huising, Hiromitsu Nakauchi, Pablo Juan Ross

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
JournalTransgenic Research
DOIs
StateAccepted/In press - Jan 1 2018

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
embryo implantation
Mosaicism
mutagenesis
biopsy
Sheep
embryo (animal)
mutation
Biopsy
sheep
Mutation
Embryonic Structures
viability
screening
gene targeting
disease diagnosis
embryo transfer
pancreas
Polymerase Chain Reaction
gel electrophoresis

Keywords

  • Biopsy
  • Gene-editing
  • Livestock
  • Mosaic
  • Ovine

ASJC Scopus subject areas

  • Biotechnology
  • Animal Science and Zoology
  • Genetics
  • Agronomy and Crop Science

Cite this

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep. / Vilarino, Marcela; Suchy, Fabian Patrik; Rashid, Sheikh Tamir; Lindsay, Helen; Reyes, Juan; McNabb, Bret; van der Meulen, Talitha; Huising, Mark O.; Nakauchi, Hiromitsu; Ross, Pablo Juan.

In: Transgenic Research, 01.01.2018.

Research output: Contribution to journalArticle

Vilarino, Marcela ; Suchy, Fabian Patrik ; Rashid, Sheikh Tamir ; Lindsay, Helen ; Reyes, Juan ; McNabb, Bret ; van der Meulen, Talitha ; Huising, Mark O. ; Nakauchi, Hiromitsu ; Ross, Pablo Juan. / Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep. In: Transgenic Research. 2018.
@article{c50a6d04c7754ce395d2636ac8f493f9,
title = "Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep",
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.",
keywords = "Biopsy, Gene-editing, Livestock, Mosaic, Ovine",
author = "Marcela Vilarino and Suchy, {Fabian Patrik} and Rashid, {Sheikh Tamir} and Helen Lindsay and Juan Reyes and Bret McNabb and {van der Meulen}, Talitha and Huising, {Mark O.} and Hiromitsu Nakauchi and Ross, {Pablo Juan}",
year = "2018",
month = "1",
day = "1",
doi = "10.1007/s11248-018-0094-x",
language = "English (US)",
journal = "Transgenic Research",
issn = "0962-8819",
publisher = "Springer Netherlands",

}

TY - JOUR

T1 - Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep

AU - Vilarino, Marcela

AU - Suchy, Fabian Patrik

AU - Rashid, Sheikh Tamir

AU - Lindsay, Helen

AU - Reyes, Juan

AU - McNabb, Bret

AU - van der Meulen, Talitha

AU - Huising, Mark O.

AU - Nakauchi, Hiromitsu

AU - Ross, Pablo Juan

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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.

AB - 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.

KW - Biopsy

KW - Gene-editing

KW - Livestock

KW - Mosaic

KW - Ovine

UR - http://www.scopus.com/inward/record.url?scp=85054519418&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85054519418&partnerID=8YFLogxK

U2 - 10.1007/s11248-018-0094-x

DO - 10.1007/s11248-018-0094-x

M3 - Article

C2 - 30284144

AN - SCOPUS:85054519418

JO - Transgenic Research

JF - Transgenic Research

SN - 0962-8819

ER -