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Development of thyroid deiodinase knockout Xenopus tropicalis using CRISPR/Cas12a gene editing towards establishment of an amphibian Adverse Outcome Pathway.
Citation:
Mayasich, S., P. Degoey, J. Haselman, AND S. Degitz. Development of thyroid deiodinase knockout Xenopus tropicalis using CRISPR/Cas12a gene editing towards establishment of an amphibian Adverse Outcome Pathway. SETAC North America, Fort Worth, TX, November 15 - 19, 2020. https://doi.org/10.23645/epacomptox.13052954
Impact/Purpose:
Amphibians are sensitive indicator species of ecosystem health. Thyroid deiodinase enzymes are important for amphibian development and metamorphosis. Our goal is to disrupt the deiodinase genes in Xenopus tropicalis using the CRISPR/Cas gene editing technique to characterize effects and contribute to Adverse Outcome Pathway (AOP) development. The poster will be presented to environmental scientists. 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. This method is also part of an approach to greatly reduce the use of live animals for chemical toxicity screening.
Description:
Three iodothyronine deiodinase enzymes (dio1, 2, and 3) are present in vertebrate animals including amphibians, and are involved in balancing the thyroid hormone system in synchronized developmental and metamorphic processes. Our ultimate goal is to knock out the amphibian deiodinase genes to characterize effects and to contribute to amphibian thyroid adverse outcome pathway (AOP) development. We chose the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a system because it provided advantages including DNA cleavage locations targeting the catalytic site, and slightly better efficiency compared to Cas9. Experiments separately targeting the X. 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 extraction and polymerase chain reaction (PCR) genotyping analysis method was developed to screen more than 200 injected tadpoles to distinguish “crispant” tadpoles with gene disruption from RNP-injected “wildtype” tadpoles displaying no gene disruption (no difference from uninjected controls). Microinjection of the respective RNPs resulted in dio2 gene disruption in more than 60% of tadpoles, and dio3 gene disruption in 50% of tadpoles screened at 3 weeks post-injection. The dio2 crispant tadpoles showed a number of changes consistent with complete or partial disruption of the metamorphic process. Dio2 crispants reached Nieuwkoop and Faber developmental stage 62 earlier than controls and we observed significant abnormalities which included delayed limb development and accelerated asynchronous metamorphosis. In dio3 crispants a high level of mortality was observed (83%) which began to occur when the tadpoles reached the prometamorphosis stages. Tadpoles evaluated shortly after death were observed to have undergone precocious metamorphosis. We observed tail and gill resorption at early stages of prometamorphosis which was likely the cause of that mortality. This work-in-progress provides preliminary support for deiodinase disruption as a molecular initiating event in the amphibian thyroid AOP, and a link with deiodinase-inhibiting chemicals such as those we identified previously in a X. laevis recombinant dio3 enzyme in vitro screening of more than 350 chemicals. The combined in vitro chemical screening and gene knockout approaches can also greatly reduce the use of live animals for chemical toxicity screening.
URLs/Downloads:
DOI: Development of thyroid deiodinase knockout Xenopus tropicalis using CRISPR/Cas12a gene editing towards establishment of an amphibian Adverse Outcome Pathway.