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

Abstract

The domestic ferret (Mustela putorius furo) 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 ROSA26 “safe harbor” locus in ferret zygotes and created transgenic animals expressing a dual-fluorescent Cre-reporter system flanked by PhiC31 and Bxb1 integrase attP sites. Out of 151 zygotes injected with circular transgene-containing plasmid and Cas9 protein loaded with the ROSA26 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 ROSA26-CAG-LoxPtdTomatoStopLoxPEGFP (ROSA-TG) Cre-reporter was confirmed in primary cells following Cre expression. The Phi31 and Bxb1 integrase attP sites flanking the transgene will also enable rapid directional insertion of any transgene without a size limitation at the ROSA26 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.

The Ferret as a Model System for Neocortex Development and Evolution

“…Fifth, transgenic ferrets are now available ( Johnson et al, 2018 ; Yu et al, 2019 ). Moreover [combining the steps 6–9 from Matthews and Vosshall (2020) ] genome editing ( Shinmyo et al, 2017 ), routine gene knock-down ( Kostic et al, 2019 ) and knock-out ( Güven et al, 2020 ; Xing et al, 2020 ), introduction of precise mutation ( Yu et al, 2019 ), optogenetics ( Roy et al, 2016 ) and other genetic tools important for neuroscience have all been introduced to the ferrets. Finally, interesting questions that can be addressed using the ferret as the model organism are not lacking and include, but are not limited to, the considerations we discuss below.…”

“…The knockout was achieved by Transcription Activator Like Effector Nucleases (TALEN)-mediated genome editing. A year later, Yu et al (2019) reported the creation of a transgenic ferret line expressing a dual-fluorescent Cre-reporter system inserted into the ferret ROSA26 intron 1. In this case CRISPR/Cas9-based genome editing was employed.…”

“…Its genome has been sequenced ( Peng et al, 2014 ) and several different methods that enable acute genetic manipulation in the embryonic and postnatal brain are available ( Borrell, 2010 ; Kawasaki et al, 2012 ). Finally, ferret transgenesis has recently been established ( Johnson et al, 2018 ; Yu et al, 2019 ). In this review we discuss those features of the ferret neurodevelopment that make this animal an important model system for the study of cell biology of neural progenitors, neuronal differentiation and migration, mechanisms underlying gyrification, development of the sensory systems and human pathologies pertinent to the central nervous system.…”

Improvements in Gene Editing Technology Boost Its Applications in Livestock

“…Often these approaches aim to accomplish specific targeted integration of genes of interest into what is known as safe harbor’ genes, such as Rosa26, adeno-associated virus integration site 1 (AAVS1), and H11 ( Ruan et al, 2015 ; Wu et al, 2016 ; Xie et al, 2017 ). These sites in the genome are able to accommodate the transgene integration that ensures its high transcriptional activity in embryonic and adult tissues, and does not suppress critical endogenous genes ( Ruan et al, 2015 ; Oceguera-Yanez et al, 2016 ; Weber et al, 2016 ; Wu et al, 2016 ; Yu et al, 2019 ; Kelly et al, 2020 ).…”

“…Zygotes ( n = 151) were microinjected with the plasmid and CRISPR/Cas9 RNP. Five out of 23 offspring exhibited the reporter expression ( Yu et al, 2019 ). Therefore, HITI method offers a great enhancement over the other methods as it takes advantage of NHEJ for gene insertion.…”