Optimizing hfCas13X for mRNA depletion in Drosophila germ granules

Abstract

Pole cell development in Drosophila is key to establishing the germ line. Within pole cells are ribonucleoprotein granules, known as germ granules, that contain key maternal determinants for pole cell formation, including nanos and pgc. It is not fully understood as to the role of every maternally provided mRNA that is enriched within the germ granules. RNA knockdown is a method by which researchers can understand the function of an mRNA. However current RNA knockdown tools are limited and cannot adequately be subcellularly localized. The CRISPR/Cas13 system is a method by which RNA can be subcellularly depleted, but its collateral activity once activated makes phenotype attribution difficult. This paper worked to optimize Cas13 for usage in subcellular knockdown of mRNA within the Drosophila germ granules. In particular, this paper looked at Gwl, a protein kinase previously reported to be found in the germ granules. I showed that gwl mRNA is expressed broadly across the early Drosophila embryo with weak but detectable enrichment in the posterior pole. I then laid the foundation for knockdown of gwl by selecting hfCas13X, a modified version of Cas13X that has been shown to have reduced collateral activity in-vitro, and testing translational control and localization by appending nanos and pgc regulatory sequences to the hfCas13X sequence. Despite generating flies with this transgene, hfCas13X was not expressed. I was able to successfully design crRNA arrays target gwl and nanos and generate transgenic fly stocks for both of these constructs, which can be used in future experiments with hfCas13X.