Leveraging CRISPR-Cas13d in an inducible knockdown system to interrogateDrosophilagerm granule mRNAs

Abstract

ABSTRACTRibonucleoprotein (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 inDrosophila, 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 (nos)andsarah (sra), whose abundance in germ granules differs. Because Cas13 has bothcisandtranscleavage 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 abundantnosgerm granule transcripts. Off-target RNA knockdown is less potent when the system is used for less abundantsratranscripts. Whensrais knocked down in germ granules, we observe defective primordial germ cell migration, and an increase in the calcium indicator GCaMP at the posterior, consistent withsraencoding a negative regulator of calcium signaling. In sum, we report anin vivoCas13-based system for subcellular knockdown, evaluate its feasibility, and uncover a novel function forsragerm granule transcripts in promoting germline development.