Highly Efficient Transgenesis in Ferrets Using CRISPR/Cas9-Mediated Homology-Independent Insertion at the ROSA26 Locus

<jats:title>Abstract</jats:title><jats:p>The domestic ferret (<jats:italic>Mustela putorius furo</jats:italic>) has proven to be a useful species for modeling human genetic and infectious diseases of the lung and brain. However, biomedical research in ferrets has been hindered by the lack of rapid and cost-effective methods for genome engineering. Here, we utilized CRISPR/Cas9-mediated, homology-independent insertion at the <jats:italic>ROSA26</jats:italic> “safe harbor” locus in ferret zygotes and created transgenic animals expressing a dual-fluorescent Cre-reporter system flanked by PhiC31 and Bxb1 integrase <jats:italic>att</jats:italic>P sites. Out of 151 zygotes injected with circular transgene-containing plasmid and Cas9 protein loaded with the <jats:italic>ROSA26</jats:italic> intron-1 sgRNA, there were 23 births of which 5 had targeted integration events (22% efficiency). The encoded tdTomato transgene was highly expressed in all tissues evaluated. Targeted integration was verified by PCR analyses, Southern blot, and germ-line transmission. Function of the <jats:italic>ROSA26</jats:italic>-CAG-<jats:sup>LoxP</jats:sup><jats:italic>tdTomato</jats:italic><jats:sup>StopLoxP</jats:sup><jats:italic>EGFP</jats:italic> (<jats:italic>ROSA</jats:italic>-TG) Cre-reporter was confirmed in primary cells following Cre expression. The Phi31 and Bxb1 integrase <jats:italic>att</jats:italic>P sites flanking the transgene will also enable rapid directional insertion of any transgene without a size limitation at the <jats:italic>ROSA26</jats:italic> locus. These methods and the model generated will greatly enhance biomedical research involving lineage tracing, the evaluation of stem cell therapy, and transgenesis in ferret models of human disease.</jats:p>