Engineering Cas7-11 for Subcellular RNA Knockdown in the Drosophila Germ Plasm

Abstract

Germ cell formation is essential for sexual reproduction. In many animals, this process depends on the germ plasm, which is a specialized maternally inherited cytoplasm that directs germ cell fate. In Drosophila embryos, the germ plasm is localized to the posterior pole and is organized into germ granules that concentrate maternal mRNAs needed for germ cell development. Although a few of these transcripts, such as nanos and pgc, have known functions in germ cell specification, the roles of the vast majority remain untested because mutations in germ plasm components disrupt germ plasm assembly altogether, and RNA interference lacks the spatial precision to deplete transcripts specifically within the germ plasm. To overcome these limitations, I aimed to develop a subcellular RNA knockdown system based on Cas7-11 a CRISPR endonuclease that causes degradation of target RNA without the collateral cleavage activity associated with existing Cas13-based systems. To restrict Cas7-11 activity to the germ plasm, I fused it to the pgc 3'UTR and the nanos translational control element, regulatory sequences that together localize the mRNA to the posterior pole and silence translation among unlocalized copies. Immunofluorescence confirms that Cas7-11 protein accumulates in the embryonic germ plasm and is inherited by pole cells. Single-molecule fluorescence in situ hybridization reveals that the crRNA-directed Cas7-11 effect can reduce nanos transcript levels at the posterior pole. nanos knockdown does not diminish levels of non-target germ granule mRNAs, which is consistent with the expected absence of collateral cleavage. Together, these findings demonstrate that Cas7-11 can achieve targeted RNA depletion within a defined subcellular compartment, providing possibilities for systematic functional analysis of individual germ granule mRNAs in the process of germ cell development.