This proposal supports a project to construct over 100 different Xenopus mutants in key genes related to human disease. The proposed studies will use CRISPR/Cas and TALEN gene editing to create precise models of human disease in the amphibian Xenopus. After a survey of the Xenopus community, there are over 150 different mutants requested to support biomedical research in Xenopus. The current studies are focused on developing Xenopus models of many different diseases for the entire Xenopus community, and each mutant will be developed in close coordination with individual researchers. There are three main aims to this proposal. First, mutations in key genes will be produced in either Xenopus tropicalis or Xenopus laevis using CRISPR/Cas or TALEN gene editing methods. Second, we propose to develop the use of oocyte host transfer for the generation of mutants using CRISPR/Cas and TALEN methods; this will increase the efficiency with which mutations are induced prior to first embryonic cell division and to allow for the creation of uniform heterozygous mutations by limiting Cas9 activity only in the oocyte. Third, we will optimize knock-in strategies with CRISPR/Cas and/or TALEN. All of these aims will help enhance the utilization of CRISPR/Cas and TALEN gene editing methods in the Xenopus model system.
Research using the amphibian Xenopus, because of unique advantages as an experimental system, has revealed key insights in many domains of biomedical research, including cell biology, development, neurobiology, physiology and signal transduction. This proposal seeks to generate over 100 different Xenopus mutants in key genes related to human diseases using new technology, which will help provide significant insights about the causes of these diseases.
