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Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus

View ORCID ProfileYuji Matsuoka, View ORCID ProfileTaro Nakamura, View ORCID ProfileTakahito Watanabe, View ORCID ProfileAusten A. Barnett, View ORCID ProfileSumihare Noji, View ORCID ProfileTaro Mito, View ORCID ProfileCassandra G. Extavour
doi: https://doi.org/10.1101/2021.05.10.441399
Yuji Matsuoka
1Department of Life Systems, Institute of Technology and Science, the University of Tokushima Graduate School, 201 Minami-Jyosanjima-cho, Tokushima City, 770-8506, Japan
3Department of Biological Sciences, National University of Singapore, 14 Science Drive 4 117543, Singapore
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  • For correspondence: dbsyuji@nus.edu.sg taro@nibb.ac.jp mito.taro@tokushima-u.ac.jp extavour@oeb.harvard.edu
Taro Nakamura
2Department of Organismic and Evolutionary Biology, 16 Divinity Avenue, Cambridge MA 02138, USA
4National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Aichi, Japan
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Takahito Watanabe
1Department of Life Systems, Institute of Technology and Science, the University of Tokushima Graduate School, 201 Minami-Jyosanjima-cho, Tokushima City, 770-8506, Japan
5Bio-Innovation Research Center, Tokushima University, 2272-2 Ishii, Ishii-cho, Myozai-gun, Tokushima 779-3233, Japan
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Austen A. Barnett
2Department of Organismic and Evolutionary Biology, 16 Divinity Avenue, Cambridge MA 02138, USA
6DeSales University, 2755 Station Avenue, Center Valley, PA 18034, USA
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Sumihare Noji
7Tokushima University, 2-14 Shinkura-cho, Tokushima City, 770-8501, Japan
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Taro Mito
1Department of Life Systems, Institute of Technology and Science, the University of Tokushima Graduate School, 201 Minami-Jyosanjima-cho, Tokushima City, 770-8506, Japan
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  • For correspondence: dbsyuji@nus.edu.sg taro@nibb.ac.jp mito.taro@tokushima-u.ac.jp extavour@oeb.harvard.edu
Cassandra G. Extavour
2Department of Organismic and Evolutionary Biology, 16 Divinity Avenue, Cambridge MA 02138, USA
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  • For correspondence: dbsyuji@nus.edu.sg taro@nibb.ac.jp mito.taro@tokushima-u.ac.jp extavour@oeb.harvard.edu
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Abstract

Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yields viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted May 10, 2021.
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Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus
Yuji Matsuoka, Taro Nakamura, Takahito Watanabe, Austen A. Barnett, Sumihare Noji, Taro Mito, Cassandra G. Extavour
bioRxiv 2021.05.10.441399; doi: https://doi.org/10.1101/2021.05.10.441399
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Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus
Yuji Matsuoka, Taro Nakamura, Takahito Watanabe, Austen A. Barnett, Sumihare Noji, Taro Mito, Cassandra G. Extavour
bioRxiv 2021.05.10.441399; doi: https://doi.org/10.1101/2021.05.10.441399

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