A selection-based knockout approach for a choanoflagellate reveals regulation of multicellular development by Hippo signaling

Choanoflagellates, the closest living relatives of animals, provide crucial insights into animal origins. The multicellular choanoflagellate Salpingoeca rosetta can be genetically modified, but existing knockout (KO) pipelines are time consuming and have variable efficiency. Here, we present a fast and robust KO method for S. rosetta. We use CRISPR-Cas9 to inactivate target genes by interrupting, or fully replacing, their coding sequence with a selectable antibiotic resistance cassette. We inactivated three known S. rosetta multicellular developmental regulators (rosetteless, couscous, and jumble) and two homologs of Hippo pathway genes that control multicellular size in animals (warts and yorkie). Interestingly, warts-KO rosettes were consistently larger than their wild-type counterparts. RNA sequencing revealed that Warts and Yorkie regulated several extracellular matrix genes involved in multicellularity (including couscous), suggesting that Hippo signaling regulates multicellular size in choanoflagellates by modulating matrix secretion. We discuss the potential of our method to accelerate choanoflagellate functional genetics.