Crispr/Cas9 Gene Editing Targeted to an Intron of a Novel Isoform of the β-Subunit of Thyroid Stimulating Hormone in Peripheral Leukocytes

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Abstract Hashimoto’s disease and Graves’ disease are two forms of autoimmune thyroiditis (AIT). Although various treatments protocols are available for each disease, the specific events involved in the induction of AIT remain largely unknown. We identified a novel splice variant of the β-subunit of thyroid stimulating hormone (TSHβ) in humans and mice, referred to as TSHβv, that is produced almost exclusively by bone marrow cells and peripheral leukocytes. In mice, pituitary-derived TSHβ uses exon 4 and exon 5 to code for the TSHβ polypeptide, whereas TSHβv uses exon 5 and the 3′ end of intron 4. We recently demonstrated that during viral and bacterial infection, peripheral leukocytes traffic to the thyroid and produce large amounts of TSHβv intrathyroidally. We have speculated that although this may have beneficial physiological effects for the host, it also may be a factor in the initiation and/or perpetuation of AIT. To explore this, we made Crispr/Cas9 knockout mice in which the production of immune system TSHβv but not pituitary TSHβ is disrupted by insertion of a stop codon in the terminal portion of intron 4. Of 24 founder mice, 3 had gene editing in intron 4 as determined by PCR and sequence analyses. Those animals were back-crossed with wildtype mice to get F1 heterozygous colonies and clean genomic DNA with deletions at the stop codon insertion site. F1 offspring from two of the founder mice lacked the gene-edited site in intron 4. However, 6 offspring from the third founder animal had the expected editing in intron 4. Those animals are being bred to obtain homozygous mice for experimental use. To our knowledge, this is one of the few examples of gene disruption targeted to an intron with potential functional deletion of a novel isoform.