Open AccessZygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs
Author(s) -
Kristin M. Whitworth,
Joshua A. Benne,
Lee D. Spate,
Shaun Murphy,
Melissa Samuel,
Clifton N. Murphy,
Jürgen A. Richt,
Eric Walters,
Randall S. Prather,
Kevin D. Wells
Publication year - 2016
Publication title -
transgenic research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.724
H-Index - 83
eISSN - 1573-9368
pISSN - 0962-8819
DOI - 10.1007/s11248-016-9989-6
Subject(s) - biology , crispr , blastocyst , zygote , cas9 , gene , rna , microbiology and biotechnology , genetics , embryonic stem cell , embryogenesis , guide rna , embryo , andrology , medicine
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.