Multigenerational laboratory culture of pelagic ctenophores and CRISPR-Cas9 genome editing in the lobate Mnemiopsis leidyi.

Despite long-standing experimental interest in ctenophores due to their unique biology, ecological influence and evolutionary status, previous work has largely been constrained by the periodic seasonal availability of wild-caught animals and difficulty in reliably closing the life cycle. To address this problem, we have developed straightforward protocols that can be easily implemented to establish long-term multigenerational cultures for biological experimentation in the laboratory. In this protocol, we describe the continuous culture of the Atlantic lobate ctenophore Mnemiopsis leidyi. A rapid 3-week egg-to-egg generation time makes Mnemiopsis suitable for a wide range of experimental genetic, cellular, embryological, physiological, developmental, ecological and evolutionary studies. We provide recommendations for general husbandry to close the life cycle of Mnemiopsis in the laboratory, including feeding requirements, light-induced spawning, collection of embryos and rearing of juveniles to adults. These protocols have been successfully applied to maintain long-term multigenerational cultures of several species of pelagic ctenophores, and can be utilized by laboratories lacking easy access to the ocean. We also provide protocols for targeted genome editing via microinjection with CRISPR-Cas9 that can be completed within ~2 weeks, including single-guide RNA synthesis, early embryo microinjection, phenotype assessment and sequence validation of genome edits. These protocols provide a foundation for using Mnemiopsis as a model organism for functional genomic analyses in ctenophores.