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. 2022;2450:437–465. doi: 10.1007/978-1-0716-2172-1_23

Manipulation of Gene Activity in the Regenerative Model Sea Anemone, Nematostella vectensis.

Eric M Hill, Cheng-Yi Chen, Florencia Del Viso, Lacey R Ellington, Shuonan He, Ahmet Karabulut, Ariel Paulson, Matthew C Gibson
PMCID: PMC9761902  PMID: 35359322

Abstract

With a surprisingly complex genome and an ever-expanding genetic toolkit, the sea anemone Nematostella vectensis has become a powerful model system for the study of both development and whole-body regeneration. Here we provide the most current protocols for short-hairpin RNA (shRNA )-mediated gene knockdown and CRISPR/Cas9-targeted mutagenesis in this system. We further show that a simple Klenow reaction followed by in vitro transcription allows for the production of gene-specific shRNAs and single guide RNAs (sgRNAs) in a fast, affordable, and readily scalable manner. Together, shRNA knockdown and CRISPR/Cas9-targeted mutagenesis allow for rapid screens of gene function as well as the production of stable mutant lines that enable functional genetic analysis throughout the Nematostella life cycle.

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