Zygote injection of CRISPR-Cas9 Modifies the Target Gene without Unwanted Effects in Pigs

The CRISPR-Cas9 genome editing tool has been instrumental in creating genetically modified pigs for use as biomedical or agricultural models. However, it is still unknown if the tool has an effect on the development of pig embryos. University of Missouri researchers led by Kristin Whitworth now aim to determine if DNA editing can result in delays in development to the blastocyst stage or in skews in the sex ratio.

Six DNA templates that were designed to express guide RNAs that target the transmembrane protease, serine S1, member 2 (TMPRSS2) gene were 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. Embryos were then transferred into recipient gilts.

Analysis found that the rate of blastocyst development was not significantly different between CRISPR-injected embryos and the non-injected controls at day 5, 6 or 7. Injection of CRISPR resulted in detectable mutations in 92–100% of the embryos analyzed. There was also no difference in the number of edits or sex ratio of male to female embryos when compared to the controls.

There were 12 resulting piglets and all had biallelic edits of TMRPSS2. Zygote injection with CRISPR-Cas9 proves to be an efficient tool to genetically modify pig embryos without any unwanted effects.

For more on this study, read the article in Transgenic Research.