title: Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs.
abstract: The CRISPR/Cas9 genome editing tool has increased the efficiency of creating genetically modified pigs for use as biomedical or agricultural models. The objectives were to determine if DNA editing resulted in a delay in development to the blastocyst stage or in a skewing of the sex ratio. Six DNA templates (gBlocks) that were designed to express guide RNAs that target the transmembrane protease, serine S1, member 2 (TMPRSS2) gene were in vitro transcribed. Pairs of CRISPR guide RNAs that flanked the start codon and polyadenylated Cas9 were co-injected into the cytoplasm of zygotes and cultured in vitro to the blastocyst stage. Blastocysts were collected as they formed on days 5, 6 or 7. PCR was performed to determine genotype and sex of each embryo. Separately, embryos were surgically transferred into recipient gilts on day 4 of estrus. The rate of blastocyst development was not significantly different between CRISPR injection embryos or the non-injected controls at day 5, 6 or 7 (p = 0.36, 0.09, 0.63, respectively). Injection of three CRISPR sets of guides resulted in a detectable INDEL in 92-100 % of the embryos analyzed. There was not a difference in the number of edits or sex ratio of male to female embryos when compared between days 5, 6 and 7 to the controls (p > 0.22, >0.85). There were 12 resulting piglets and all 12 had biallelic edits of TMRPSS2. Zygote injection with CRISPR/Cas9 continues to be a highly efficient tool to genetically modify pig embryos.
authors.affiliation: Division of Animal Sciences, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.|National Swine Research and Resource Center, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Whitworth
authors.affiliation: Division of Animal Sciences, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.|National Swine Research and Resource Center, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Benne
authors.affiliation: Division of Animal Sciences, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.|National Swine Research and Resource Center, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.
authors.forename: Lee D
authors.initials: LD
authors.lastname: Spate
authors.affiliation: Division of Animal Sciences, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Murphy
authors.affiliation: Division of Animal Sciences, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.|National Swine Research and Resource Center, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Samuel
authors.affiliation: Division of Animal Sciences, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.|National Swine Research and Resource Center, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.
authors.forename: Clifton N
authors.initials: CN
authors.lastname: Murphy
authors.affiliation: College of Veterinary Medicine, Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, USA.
authors.forename: Jürgen A
authors.initials: JA
authors.lastname: Richt
authors.affiliation: National Swine Research and Resource Center, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Walters
authors.affiliation: Division of Animal Sciences, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA. PratherR@Missouri.edu.|National Swine Research and Resource Center, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA. PratherR@Missouri.edu.
authors.forename: Randall S
authors.initials: RS
authors.lastname: Prather
authors.affiliation: Division of Animal Sciences, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.|National Swine Research and Resource Center, University of Missouri, 920 East Campus Dr., E125D ASRC, Columbia, MO, 65211, USA.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Wells
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000254:growth & development; D001755:Blastocyst / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics; D047108:Embryonic Development / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012744:Sex Ratio; D013552:Swine / Q000235:genetics* / Q000254:growth & development; D015053:Zygote / Q000254:growth & development*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR/Cas9; DNA editing; Zygote injection; gBlock