DOI: 10.25338/b88k97
title: Edits of AR in porcine blastocysts after CRISPR Cas9 editing of zygotes

description: The CRISPR Cas9 technology offers an opportunity to evaluate physiological
 responses to deletion of individual genes in livestock species, expanding
 our understanding of animal biology. Testing efficacy of individual guides
 used for gene deletion is desirable before the time-consuming embryo
 transfers planned to generate the desired model animals. These experiments
 were designed to test efficacy of guides to delete expression of the
 androgen receptor (AR) in pig zygotes and to characterize the resulting
 deletions. Two guides targeting exon 2 (Ensembl Sus Scrofa 11.1) were
 designed. Following electroporation of these guides into in vitro
 generated porcine zygotes, embryos were cultured to the blastocyst stage
 and the DNA sequenced. Over 80% of blastocysts produced following
 electroporation of the two guides targeting exon 2 had deletions.
 Deletions were biallelic. Although blastocysts might contain edits from
 only one of the guides, the majority of blastocysts had large deletions
 resulting from the combined effect of both guides.
description: Cumulus oocyte complexes were aspirated from 3-5 mm follicles on
 pig ovaries obtained from a commercial abbatoir, rinsed and matured in
 vitro for 40-44 hours at 38.5<sup> o </sup>C. The 100 μL drop
 containing oocytes and approximately 1000 washed, partially capacitated
 sperm/oocyte was incubated for 5-6 hours in 5% CO<sub>2</sub>
 in air. Zygotes were electroporated in Opti-MEM™ (Gibco) with the CRISPR
 guide and Cas9 protein 9 hours after insemination. Following culture of
 electroporated zygotes to the blastocyst stage, individual blastocysts
 were lysed in 10 µL of QE09050 buffer (Biosearch
 Technologies, UK), vortexed, centrifuged, and incubated at 65°C (6
 minutes) and 98°C (2 minutes)
 followed by storage at -17<sup>o </sup>C. DNA was amplified
 during nested rounds (2) of PCR, the PCR products were separated on a 0.8%
 agarose gel, bands excised, DNA was extracted (QIAquick Gel Extraction
 Kit, Qiagen #28704) and submitted for sequencing. Sequences were aligned
 with SnapGeneâ using the Sus Scrofa 11.1 genome. The sequences around the target sites of the two CRISPR guides from 9 representative blastocysts are presented in Figure 1. With electroporation with the combination of the two CRISPR guides targeting exon 2 of the porcine AR (targets outlined in red in the reference sequence), the vast majority of blastocysts have large deletions and a blastocyst has a 6 bp deletion. The last edit might only eliminate two amino acids in the transcript or potentially alter a third amino acid as well. The diversity of edits in the blastocysts with the majority being large deletions accurately forecasts the diversity of edits in fetuses, again with frequent large deletions observed.  
description: Data file is in a Word document. This can be opened with the
 open-source program Writer.
description: # CRISPR Cas9 edits of AR in porcine blastocysts *** Dataset contains the
 partial sequence of the AR in porcine blastocysts. The two CRISPR guides
 are identified by the red circle and the unedited porcine sequence is
 repeated above each edited sequence. The sequences of edited blastocysts
 in the regions of interest (target sites of guides) are provided. Oocytes
 were aspirated from porcine follicles, in vitro matured and fertilized in
 vitro. Zygotes were electroporated with the CRISPR Cas9 guides and
 cultured to the blastocyst stage. Blastocysts were then processed for DNA
 sequencing and the sequences for representative blastocysts are provided
 in Figure 1. ## Description of the data and file structure The figure
 indicates the edited sequence for each of the blastocysts, all of which
 contained biallelic edits ## Sharing/Access information No additional
 information.