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DEVELOPMENT OF TYPE 2 AND TYPE 3 IODOTHYRONINE DEIODINASE KNOCKOUT XENOPUS TROPICALIS USING CRISPR/CAS12A GENE EDITING
Citation:
Mayasich, S., P. Degoey, J. Haselman, AND S. Degitz. DEVELOPMENT OF TYPE 2 AND TYPE 3 IODOTHYRONINE DEIODINASE KNOCKOUT XENOPUS TROPICALIS USING CRISPR/CAS12A GENE EDITING. North American Society for Comparative Endocrinology (NASCE) 6th biennial meeting, Duluth, MN, May 25 - 27, 2021. https://doi.org/10.23645/epacomptox.14627562
Impact/Purpose:
Amphibians are sensitive indicator species of ecosystem health. Thyroid deiodinase enzymes are important for amphibian development and metamorphosis. Our goal is to characterize effects on metamorphosis of deiodinase activity loss in Xenopus tropicalis using the CRISPR/Cas gene editing system. The data will be presented to scientists who study comparative endocrinology. Our preliminary results indicate that abnormal metamorphosis is an adverse outcome caused by deiodinase disruption, demonstrating how amphibians might be affected by deiodinase-inhibiting chemicals in the environment.
Description:
Thyroid-mediated metamorphosis of amphibian tissues and organs is regulated in part by the activity of three iodothyronine deiodinase enzymes (dio1, 2, and 3) that catalyze tissue-localized metabolic activation/deactivation of thyroid hormones (THs) by removal of iodine. To understand potential apical effects of specific deiodinase activity loss on metamorphosis, we utilized a CRISPR/Cas12a system to induce DNA cleavage in the dio genes at locations near where the catalytic sites would occur in the translated proteins. Experiments separately targeting the Xenopus tropicalis dio2 and dio3 genes were initiated by microinjecting zygotes with gene-specific guide-RNA/Cas12a ribonucleo-protein (RNP) complexes. A rapid, non-invasive swab DNA collection and PCR genotyping method was developed to screen >200 injected tadpoles to separate gene disrupted “crispants” from wildtype (WT) tadpoles. Putative mutations occurred at a rate of >60% in dio2-RNP-injected tadpoles, and 50% in dio3-RNP-injected tadpoles screened at 3 weeks post-injection. Dio2 crispants reached Nieuwkoop and Faber (NF) developmental stage 62 earlier than controls and abnormalities included delayed limb development and asynchronous metamorphosis. In dio3 crispants a high level of mortality (83%) was observed beginning as the tadpoles reached prometamorphic stages. Tadpoles evaluated shortly after death displayed precocious tail and gill resorption at early stages of prometamorphosis, which suggests suffocation was the likely cause of death. Surviving crispant founder (F0) animals were recently bred (F0×F0, and F0×WT) and spawns are currently being evaluated for germline transmission of mutant genes. Homozygous mutants will be utilized to characterize TH profiles and definitive apical outcomes. The contents of this abstract neither constitute, nor necessarily reflect, US EPA policy.
URLs/Downloads:
DOI: DEVELOPMENT OF TYPE 2 AND TYPE 3 IODOTHYRONINE DEIODINASE KNOCKOUT XENOPUS TROPICALIS USING CRISPR/CAS12A GENE EDITING
ORD-042279 MAYASICH.PDF (PDF, NA pp, 1987.965 KB, about PDF)