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Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex

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Title: Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex
Authors: Hiruta, Chizue Browse this author →KAKEN DB
Kakui, Keiichi Browse this author →KAKEN DB
Tollefsen, Knut E. Browse this author
Iguchi, Taisen Browse this author →KAKEN DB
Keywords: Branchiopoda
Cas9 RNPs
Daphnia pulex
gene disruption
gene manipulation
genome editing
targeted mutagenesis
Issue Date: Jun-2018
Publisher: John Wiley & Sons
Journal Title: Genes to cells
Volume: 23
Issue: 6
Start Page: 494
End Page: 502
Publisher DOI: 10.1111/gtc.12589
Abstract: The microcrustacean Daphnia pulex is an important model for environmental, ecological, evolutionary and developmental genomics because its adaptive life history displays plasticity in response to environmental changes. Even though the whole-genome sequence is available and omics data have actively accumulated for this species, the available tools for analyzing gene function have thus far been limited to RNAi (RNA interference) and TALEN (the transcription activator-like effector nuclease) systems. The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is thus expected to further increase the genetic tractability of D.pulex and to advance the understanding of this species. In this study, we developed a genome editing system for D.pulex using CRISPR/Cas9 ribonucleoprotein complexes (Cas9 RNPs). We first assembled a CRISPR single-guide RNA (sgRNA) specific to the Distal-less gene (Dll), which encodes a homeodomain transcription factor essential for distal limb development in invertebrates and vertebrates. Then, we injected Cas9 RNPs into eggs and evaluated its activity in vivo by a T7 endonuclease I assay. Injected embryos showed defective formation of the second antenna and disordered development of appendages, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.
Rights: This is the peer reviewed version of the following article: "Genes to Cells" Volume23, Issue6 June 2018 Pages 494-502, which has been published in final form at This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
Type: article (author version)
Appears in Collections:理学院・理学研究院 (Graduate School of Science / Faculty of Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 蛭田 千鶴江

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