CRISPR-Cas9 genome editing in Corallochytrium limacisporum,a key species for understanding animal origins.

Abstract

Microbial holozoans are the closest unicellular relatives of animals. They share a substantial gene repertoire with animals and exhibit complex life cycles. Studying these organisms is crucial for understanding the evolution of multicellularity, and significant progress has been made in uncovering key aspects of the biology of the four microbial holozoans lineages: choanoflagellates, filastereans, ichthyosporeans and corallochytreans. However, reverse genetic tools are still lacking in corallochytreans, one of the earliest-branching holozoan lineages and the only known group with both coenocytic and binary fission development. Here, we present CRISPR-Cas9-mediated gene inactivation and point mutation methodologies in the corallochytrean Corallochytrium limacisporum. As a proof of concept, we inactivated the fkb12 gene, a component of the mTOR pathway, conferring rapamycin resistance, and introduced a point mutation in sdhB, encoding a subunit of succinate dehydrogenase, conferring carboxin resistance. Our results demonstrate the presence of both non-homologous end-joining and homology-directed repair pathways in C. limacisporum and shows an editing efficiency of approximately 2%. Furthermore, simultaneous gene targeting revealed a co-editing frequency of approximately 20%. Finally, this study establishes unequivocally that C. limacisporum is haploid, making it an ideal model for genetic studies and gene editing applications to unravel the molecular mechanisms involved in animal origins.