Microinjection, gene knockdown, and CRISPR-mediated gene knock-in in the hard coral,Astrangia poculata

Abstract

AbstractCnidarians have become valuable models for understanding many aspects of developmental biology including the evolution of body plan diversity, novel cell type specification, and regeneration. Most of our understanding of gene function during early development in cnidarians comes from a small number of experimental systems including the sea anemone,Nematostella vectensis. Few molecular tools have been developed for use in hard corals, limiting our understanding of this diverse and ecologically important clade. Here, we report the development of a suite of tools for manipulating and analyzing gene expression during early development in the northern star coral,Astrangia poculata. We present methods for gene knockdown using short hairpin RNAs, gene overexpression using exogenous mRNAs, and endogenous gene tagging using CRISPR-mediated gene knock-in. Combined with our ability to control spawning in the laboratory, these tools makeA. poculataa tractable experimental system for investigative studies of coral development. Further application of these tools will enable functional analyses of embryonic patterning and morphogenesis across Anthozoa and open new frontiers in coral biology research.Summary StatementThis study reports the development of the first transgenic knock-in coral, providing the opportunity to track the behavior of various cell types during early coral development.