Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna.

Akiko Naitou,Yasuhiko Kato,Takashi Nakanishi,Hajime Watanabe,Tomoaki Matsuura

doi:10.1242/bio.20149738

Abstract

ABSTRACTTranscription activator-like effector nucleases (TALENs) are artificial nucleases harboring a customizable DNA-binding domain and a FokI nuclease domain. The high specificity of the DNA-binding domain and the ease of design have enabled researchers to use TALENs for targeted mutagenesis in various organisms. Here, we report the development of TALEN-dependent targeted gene disruption in the crustacean Daphnia magna, the emerging model for ecological and toxicological genomics. First, a reporter transgene DsRed2 (EF1α-1::DsRed2) was targeted. Using the Golden Gate method with a GoldyTALEN scaffold, we constructed homodimeric and heterodimeric TALENs containing wild-type and ELD/KKR FokI domains. mRNAs that coded for either the customized homodimeric or heterodimeric TALENs were injected into one-cell-stage embryos. The high mortality of embryos injected with homodimeric TALEN mRNAs prevented us from detecting mutations. In contrast, embryos injected with heterodimeric TALEN mRNAs survived and 78%–87% of the adults lost DsRed2 fluorescence in a large portion of cells throughout the body. In addition, these adults produced non-fluorescent progenies, all of which carried mutations at the dsRed2 locus. We also tested heterodimeric TALENs targeted for the endogenous eyeless gene and found that biallelic mutations could be transmitted through germ line cells at a rate of up to 22%. Both somatic and heritable mutagenesis efficiencies of TALENs were higher than those of the CRISPR/Cas9 system that we recently developed. These results suggest that the TALEN system may efficiently induce heritable mutations into the target genes, which will further contribute to the progress of functional genomics in D. magna.

Highlights

The water flea Daphnia is a planktonic crustacean ubiquitously found in fresh water
Our results indicate that heterodimeric Transcription activator-like effector nucleases (TALENs) are useful for inducing targeted mutagenesis in D. magna
All of the customized dsr-TALEN coding sequences were linked to UTRs of D. magna vasa (Dmavas) exclusively expressed in germ cells (Sagawa et al, 2005) and the resulting chimeric RNAs were subsequently synthesized in vitro

Summary

Introduction

The water flea Daphnia is a planktonic crustacean ubiquitously found in fresh water. It occupies an important position in food webs as a link between producer algae and secondary consumers such as small fishes (Miner et al, 2012). Received 31 July 2014; Accepted 16 December 2014 anthropogenic chemicals and environmental changes (Ebert, 2005) For these reasons, Daphnia has long been used as a model animal in ecology (Lampert, 2011) and toxicology (Tatarazako and Oda, 2007). Daphnia has long been used as a model animal in ecology (Lampert, 2011) and toxicology (Tatarazako and Oda, 2007) It is an attractive model species for understanding the evolution of reproductive strategies because it usually alternates between parthenogenetic and sexual modes of reproduction with changes in environmental quality (Hebert, 1978). Targeted gene manipulation using RNA interference (RNAi) by microinjection of doublestranded RNAs (dsRNAs) into eggs has been developed to investigate the relationship between available genetic information and phenotypes in D. magna (Kato et al, 2011). To overcome the inability of this method to induce null phenotypes and the transient nature of RNAi, it was necessary to develop targeted mutagenesis

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