79 CRISPR gene editing in bovine zygotes — mutation confirmation by integration of protein expression and DNA sequencing analyses

Abstract

Confirmation of gene editing in livestock following CRISPR/Cas9 zygotic microinjection is often limited to either protein expression or gene sequencing analyses of unfixed embryos, but not both, due to the challenges of extracting DNA from fixed samples suitable for genotyping. Bovine embryos have been derived from gene-edited cell lines to screen for mutations followed by nuclear transfer, but these techniques limit embryo production efficiency, include technical challenges, and alter physiological relevance. Herein we report an integrative approach to evaluate both protein expression and the genotype of individual, fixed bovine embryos subjected to CRISPR/Cas9 microinjection. Bovine zygotes were derived from IVF and parthenogenetic activation of in vitro-matured oocytes followed by intracytoplasmic injection of CRISPR/Cas9 targeting embryonic POU5F1. Embryos were cultured for 7 days and fixed in 4% paraformaldehyde and stored at 5°C for up to 4 weeks. Fixed embryos were first subjected to immunohistochemistry to determine POU5F1 mutation success by protein expression. Binder reinforcement labels were affixed to glass slides as a substitute for adhesive spacers. Single embryos were added to 9 µL of PBS with a coverslip for imaging. Individual embryos were then recovered and placed in 10 µL of QuickExtract (Lucigen, Middleton, WI, USA) for DNA extraction by heating samples to 65°C for 6 min and 95°C for 2 min. Two rounds of PCR were applied to templates in 20-µL reactions consisting of 10 µL of GoTaq Hot Start Green Master Mix (2×; Promega, Madison, WI, USA) with the addition of 0.4 µL (10 µM) of forward and reverse primer each and 9.2 µL of template. The second PCR reaction contained 5 µL of PCR product from the first reaction, nested primers, and 4.2 µL of H₂O. The PCR conditions were |95°C, 3 min| and 35 cycles of |95°C, 30 s |56°C, 30 s|72°C, 30 s|72°C, 7 min|. The PCR product was run on an agarose gel to confirm DNA amplification of a single band, and the remainder was purified (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) and submitted (100 ng) for Sanger sequencing. The CRISPR-injected embryos were aligned to wild-type embryo sequences using SnapGene (http://www.snapgene.com/) and TIDE software (https://tide.nki.nl/). Both DNA extraction (n = 18/19) and Sanger sequencing (n = 19/23) proved highly efficient and repeatable (83 and 95%, respectively). These methods provide an efficient, cost effective, and reproducible approach for confirmation of CRISPR gene editing in individual and fixed bovine embryos by both protein expression and gene sequencing. In addition, these techniques complement the production of gene editing in livestock after zygotic CRISPR microinjection by conclusively determining mutation efficiency through both proteomic and genomic analyses.

This research was supported by a USDA NIFA AFRI ELI Postdoctoral Fellowship 2016-67012-25254 to BWD and NIFA multistate research project W3171 to PJR. BD is currently supported by a grant from the Eunice Kennedy Shiver National Institute of Child Health and Human Development of the National Institutes of Health under the award numbers T32HD087166.