The recent development of the CRISPR-Cas system has transformed many non-genetic models of cellular biology, yet the full application of its genome editing capabilities has been largely restricted to warm-blooded species and to genes which are easily amenable to current methods of CRISPR-Cas mediated gene disruption. Several species which live at lower temperatures, such as Xenopus and the marine models Loligo and Limulus, are ideal systems to model human disease, yet current methods of precise genome editing in these species are inadequate. Furthermore, due to their genomic architecture, many highly-conserved disease-causing genes are refractory to current methods of genome editing. We propose to expand the CRISPR-Cas mediated genome editing toolkit in the field of human disease modeling by driving innovation in two areas: 1) the development of a low-temperature CRISPR-Cas system capable of effecting targeted gene insertion in model species which thrive at lower temperatures, and 2) to develop a nascent system of targeted gene disruption by the application of CRISPR-Cas libraries.
The current proposal seeks to develop two new approaches to the use of CRISPR-Cas genome editing in Xenopus. If successful, this will help advance the creation of new models of human disease in Xenopus and other model systems.
