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Optimization of CRISPR/Cas9 Platform‐Mediated Gene Editing of Swine Embryos for The Creation of Novel Biomedical Swine Models

Swine are increasingly used to model complex human diseases for basic scientific discovery and for the preclinical development and validation of therapies and medical devices. The overwhelming genetic, anatomical, physiological and pathophysiological similarities to humans make swine an ideal platform for modeling the complex interplay of factors that influence human disease. The recent advances in gene editing technologies have been instrumental in opening the door to the creation of novel genetic swine models. However, in comparison to the creation of genetic rodent models, the process of creating swine models can be relatively costly, and time and labor intensive, if the techniques and experimental parameters are not optimized to allow high efficiency of edited embryos to be implanted into surrogate swine. The overarching objective of this study was to develop an optimized CRISPR/Cas9 platform for high efficiency of targeted gene editing via cytosolic microinjection of CRISPR/Cas9 components into presumptive swine embryos. We used the introduction of mutations in the neurofibromin 1 gene as the backbone for optimizing the platform. CRISPR guide RNAs (gRNA) were created for regions targeting sites both upstream and downstream of an exon within the gene for targeted cleavage by Cas9. Repair was mediated by a specific single‐stranded oligodeoxynucleotide (ssODN) template thus introducing a 2500 bp deletion. The functionality of the CRISPR/Cas9 system was validated using a plasmid‐mediated transfection of porcine kidney cells; the accuracy of editing in the target region was confirmed by Sanger sequencing. The optimization of the CRISPR/Cas9 platform was performed by the cytosolic microinjection of the editing elements into in vitro fertilized single‐celled presumptive zygotes. Varying parameters including the use of Cas9 as mRNA vs protein, Cas9 concentration, gRNA concentration, and ssoDN concentration were examined. The editing efficiencies were evaluated first with a customized multiplex nested PCR and then verified by Sanger sequencing. We found that each CRISPR concentration studied to be equally permissive of embryonic development through the blastocyst stage and that all conditions produced gene editing events at some frequency. However, the use of naked Cas9 mRNA and higher CRISPR concentrations produced greater rates of gene edit. We present our findings and optimization that allows for efficient creation of swine models.