TALENs Mediate Efficient and Heritable Mutation of Endogenous Genes in the Marine Annelid Platynereis dumerilii

Abstract

Platynereis dumerilii is a marine polychaete and an established model system for studies of evolution and development. Platynereis is also a re-emerging model for studying the molecular basis of circalunar reproductive timing: a biological phenomenon observed in many marine species. While gene expression studies have provided new insight into patterns of gene regulation, a lack of reverse genetic tools has so far limited the depth of functional analyses in this species. To address this need, we established customized transcriptional activator-like effector nucleases (TALENs) as a tool to engineer targeted modifications in Platynereis genes. By adapting a workflow of TALEN construction protocols and mutation screening approaches for use in Platynereis, we engineered frameshift mutations in three endogenous Platynereis genes. We confirmed that such mutations are heritable, demonstrating that TALENs can be used to generate homozygous knockout lines in P. dumerilii. This is the first use of TALENs for generating genetic knockout mutations in an annelid model. These tools not only open the door for detailed in vivo functional analyses, but also can facilitate further technical development, such as targeted genome editing.

Spectral Tuning of Phototaxis by a Go-Opsin in the Rhabdomeric Eyes of Platynereis

“…The Go-opsins form an ancient and poorly characterized group retained only in marine invertebrate genomes. Here, we characterize a Go-opsin from the marine annelid Platynereis dumerilii [3][4][5][12][13][14][15]. We found Go-opsin1 coexpressed with two r-opsins in depolarizing rhabdomeric photoreceptor cells in the pigmented eyes of Platynereis larvae.…”

Variation in spiralian development: insights from polychaetes

“…The application of RNAi knockdown approaches in Enchytraeus and morpholino-mediated knockdown in Helobdella demonstrates that functional genomics are possible in annelids (Takeo et al, 2010, Woodruff et al, 2007. In addition, recent development of reverse genetic tools using TALENs in Platynereis (Bannister et al, 2014), or use of CRISPR technology (Pennisi, 2013) holds great promise. The rapid advancement of sequencing technologies and drastic reduction in sequencing costs has also benefited the field.…”

Creating cell and animal models of human disease by genome editing using CRISPR/Cas9

“…Therefore, this process is considered as an applicable technique for producing model systems aiming to identify therapeutic strategies based on genome engineering. [23][24][25][26][27][28][29] The inefficiency of this method lies in the interference of epigenetic changes in the genome with the activity of TALENs. 30 The potential advantage of TALENS over ZFNs is that TALENs are not limited to binding to 9-18-bp target sites.…”

“…So far, TALENs have been used to make purposeful alterations in sequences of interest in the genome of a wide variety of organisms, including humans, rats, worms, zebrafish, pigs and yeast. Therefore, this process is considered as an applicable technique for producing model systems aiming to identify therapeutic strategies based on genome engineering . The inefficiency of this method lies in the interference of epigenetic changes in the genome with the activity of TALENs .…”