Abstract
The water flea Daphnia magna has been used as an animal model in ecology, evolution, and environmental sciences. Thanks to the recent progress in Daphnia genomics, genetic information such as the draft genome sequence and expressed sequence tags (ESTs) is now available. To investigate the relationship between phenotypes and the available genetic information about Daphnia, some gene manipulation methods have been developed. However, a technique to induce targeted mutagenesis into Daphnia genome remains elusive. To overcome this problem, we focused on an emerging genome editing technique mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system to introduce genomic mutations. In this study, we targeted a functionally conserved regulator of eye development, the eyeless gene in D. magna. When we injected Cas9 mRNAs and eyeless-targeting guide RNAs into eggs, 18–47% of the survived juveniles exhibited abnormal eye morphology. After maturation, up to 8.2% of the adults produced progenies with deformed eyes, which carried mutations in the eyeless loci. These results showed that CRISPR/Cas system could introduce heritable mutations into the endogenous eyeless gene in D. magna. This is the first report of a targeted gene knockout technique in Daphnia and will be useful in uncovering Daphnia gene functions.
Highlights
The water flea Daphnia magna is a planktonic crustacean ubiquitously found in the fresh water environment
Functional analyses of D. magna eyeless (Dma-ey) gene To utilize the ey gene as a marker gene for targeted mutagenesis, we searched for mammalian pax6 orthologs in the D. magna genome and found the ey gene in addition to its paralog, twin of eyeless gene, both of which are conserved among several arthropods [33]
Our results indicated that the eyeless gene is functionally conserved in D. magna, suggesting that it would be a useful marker gene for subsequent knockout experiments using the CRISPR/Cas system
Summary
The water flea Daphnia magna is a planktonic crustacean ubiquitously found in the fresh water environment. It has been used as a model organism in ecology and toxicology because it is sensitive to artificial chemicals and environmental changes [1]. Researchers find it interesting that Daphnia can switch their reproduction mode from asexual to sexual in response to environmental stimuli [2]. A vast amount of genetic information on Daphnia is available. To investigate the relationship between available genetic information and phenotypes, gene manipulation tools such as RNA interference (RNAi) and non-homologous integration with plasmid DNA have been developed in D. magna [5,6]. There is still no technique to induce inheritable targeted gene disruptions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
