Developing new methods: Adapting the CRISPR-Cas13 genome editing system to study germ granule mRNAs in Drosophila germ cell development and beyond

Abstract

Early in development, cells undergo a decision between germline and somatic fates. In Drosophila, the germ cell progenitors, called pole cells, form at the posterior of the embryo and contain the maternally derived germ plasm. Germ plasm contains protein-and-mRNA- rich assemblies called germ granules that promote the germ cell fate. As effective methods for knocking down transcripts locally are lacking, the functions of many germ granule mRNAs in germ cell development remain a mystery. This thesis reports a new methodology, using CRISPR-Cas13, for knocking down transcripts efficiently in a localized manner in Drosophila. I selected two germ granule transcripts, one encoding the regulator of calcineurin, Sarah (Sra) and one encoding the actin nucleation factor, Spire (Spir). I transgenically expressed CRISPR RNAs (crRNAs) targeting these transcripts in flies. I generated lines with different Cas13 transgenes that control Cas13 expression on demand. The system will be used to investigate Sra and Spir function in promoting germ cell development. This thesis also reports a Cas13-based method for live imaging of RNA dynamics. These technologies will be optimized to be relevant for other scientists. To conclude, I shift from the microscopic to the global scale to evaluate the outlook of CRISPR technologies for treating diseases.