Leveraging CRISPR-Cas13d in an inducible knockdown system to interrogate Drosophila germ granule mRNAs

ABSTRACT Ribonucleoprotein (RNP) germ granules are hallmarks of germ cells across the animal kingdom and are thought to be hubs for post-transcriptional regulation that promote formation of the germ cell precursors. While numerous RNAs are associated with germ granules in Drosophila , the functions of many in germline development are poorly understood. Current methods for RNA knockdown, such as RNAi, do not allow local depletion of transcripts such as those found in the germ granules. We leveraged CRISPR-Cas13 to create a subcellular RNA knockdown system and tested it on two mRNAs, nanos (nanos) and sarah (sra) , whose abundance in germ granules differs. Because Cas13 has both cis and trans cleavage activities, we evaluated the effect of target abundance on off-target RNA depletion. We show on and off-target RNA depletion is coupled when targeting the more abundant nanos germ granule transcripts. Off-target RNA knockdown is less potent when the system is used for less abundant sra transcripts. When sra is knocked down in germ granules, we observed an increase in the calcium indicator GCaMP at the posterior and defective primordial germ cell migration, consistent with sra encoding a negative regulator of calcium signaling. In sum, we report an in vivo Cas13-based system for subcellular knockdown, evaluate its feasibility, and uncover a novel function for sra germ granule transcripts in promoting germline development.