Single Nucleotide Genome Modifications in Oocytes

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DESCRIPTION (provided by applicant): We combine several new technologies and apply them to oocytes and embryos with the goal of quickly, efficiently, and effectively altering genomes of animals. We utilize oocytes and early embryos in a non-transgenic approach to alter specific gene function. Oocytes will be used for construction of stable transgenic lines at the F0 generation, and embryos for homozygous gene alteration for quick gene screening. This technology will open the door for the study of new organisms and to either modify a gene for functional analysis or correct a gene that is otherwise rendered sub-functional. Genome manipulation approaches have been performed mostly with cultured cells and genetically tractable embryos because of the capabilities to introduce exogenous DNA constructs. To create stable transgenic animals, however, these approaches require back-crosses to reduce genetic mosaicism or integration into the germ line and thus increase the cost of time and resources. These limitations minimize the effectiveness for broad utilization in organisms key for biological research, especially those whose genetic tractability is low. The use transcriptional activator like effector (TALE) functions linked to an enzymatic domain of DNA deaminase activity into oocytes is novel, and success of this project will allow researchers to construct gen alterations at the F0 generation. This project crafts a single nucleotide genome targeting system at a predetermined site by use of the catalytic domains of ADAR. Our recent preliminary results demonstrate DNA-deaminating activity of this domain and coupled with a demonstrable TALE-targeting mechanism increases feasibility of this project. The outcome of this project will be useful to all researchers, especially those working in genetically less-tractable organisms, and those wishing to rapidly screen gene functions without transgenic approaches. We believe this technology will remove the wording non-model organism from the genetic research vernacular, and in the future may be applicable for clinical use by correcting a point mutation related to a congenital disease state.