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Non-Transgenic Genome Modifications in a Hemimetabolous Insect Using Zinc-Finger and TAL Effector Nucleases

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Non-Transgenic Genome Modifications in a Hemimetabolous Insect Using Zinc-Finger and TAL Effector Nucleases ARTICLE Received 6 Jun 2012 | Accepted 24 Jul 2012 | Published 21 Aug 2012 DOI: 10.1038/ncomms2020 Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases Takahito Watanabe1, Hiroshi Ochiai2, Tetsushi Sakuma2, Hadley W. Horch3, Naoya Hamaguchi1, Taro Nakamura1, Tetsuya Bando1, Hideyo Ohuchi1, Takashi Yamamoto2, Sumihare Noji1 & Taro Mito1 Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations.U p to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy. 1 Department of Life Systems, Institute of Technology and Science, University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima 770-8506, Japan. 2 Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan. 3 Department of Biology and Neuroscience, Bowdoin College, Brunswick, Maine 04011, USA. Correspondence and requests for materials should be addressed to T.M. (email: [email protected]). NatURE COMMUNicatiONS | 3:1017 | DOI: 10.1038/ncomms2020 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited. All rights reserved. ARTICLE NatUre cOMMUNicatiONS | DOI: 10.1038/ncomms2020 ontinuous improvements in DNA sequencing technologies and homologous recombination mechanisms. For NHEJ-based continue to facilitate sequence analyses of complete genomes repairs of ZFN/TALEN-induced DSBs, small insertions and/or Cof numerous species. Moreover, the ability to directly and deletions (indels) are produced at the repair junction, thereby specifically manipulate a genome, for example, by generating generating mutations precisely at the cleavage site (Fig. 1a)9. specific gene knockouts, is essential for elucidating gene function ZFN-targeted mutations have been induced in several organ- on a genome-wide scale. However, the molecular tools and methods isms, including fruitflies10–12, silkworms13, zebrafish14, rats15,16, needed for gene targeting require a functional genomics approach. sea urchins17, roundworms18, frogs19 and mice20,21. More recently, Thus, these techniques have been limited to the study of certain well- TALENs have been applied to rats22, zebrafish23 and fruitflies24. In established model organisms, including yeast1, fruitflies2 and mice3, both models, mRNA was directly injected into embryos. However, although approaches in these systems are still often inefficient. As a the success of this method in engineering homozygous knock- result, a systematic, reverse genetic analysis of crickets, and perhaps out animals that exhibit null phenotypes has had limited success, other lesser-studied species, has been limited by the absence of a and has only been reported in fruitflies10,24, zebrafish14,23 and suitable genome-manipulation technique, until now. rats15,16,22. In these cases, selective breeding with animals that bear Engineered nucleases, including zinc-finger nucleases (ZFNs) specific genetic markers, or morphologically detectable phenotypes, and transcription activator-like effector nucleases (TALENs), are was performed after ZFN mutagenesis. Correspondingly, these powerful tools for genome editing based on their capacity to tar- approaches depended on genetic tractability, or the characteristics get a specific genomic locus4,5. For example, ZFNs can be generated of the phenotypes generated. To facilitate a more generally applica- by linking the nonspecific cleavage domain of the FokI restriction ble knockout analysis in various organisms, including non-model endonuclease to a DNA-binding domain composed of modular zinc- ones, a strategy to apply ZFN/TALEN-mediated mutagenesis for the finger (ZF) domains4. Each ZF domain primarily contacts three base generation of homozygous knockout animals was investigated. pairs (bps) within the major groove of DNA. However, ZFs can be The cricket, Gryllus bimaculatus (Orthoptera), is an emerging modified to recognize a broad range of DNA sequences4,6. Ideally, model system that has been used to study developmental biology a pair of ZFNs is designed for a particular target, whereby dimeri- and mechanisms of regeneration based on the success of RNA zation of the associated cleavage domains is accommodated and a interference (RNAi)-based gene-functional analyses and trans- double-stranded break (DSB) is introduced (Fig. 1a)6,7. Alterna- genic technology in this system25,26. It is possible that this cricket tively, TALENs consist of an engineered array of TAL effector repeats model could also be used to characterize the molecular mechanisms fused to the FokI cleavage domain, and can also introduce targeted relevant to human diseases, and be a model for the screening of DSBs in DNA5. The DNA-binding domains of TALEs have tandem therapeutic drugs with RNAi27. However, while the cricket is repeat units (each 33–35 aa in length), in which the nucleotide bind- currently used as a model system in the fields of neuroethol- ing preference is determined by the two adjacent amino acids, and ogy, endocrinology and behavioural ecology, the introduction of these are referred to as the repeat variable di-residue8. For both targeted and heritable mutations has remained intractable in this systems, DSBs can be restored via cellular DNA repair pathways, species. Therefore, the ability to edit the cricket genome would which include non-homologous end-joining (NHEJ) mechanisms further enhance studies performed in all of these fields. a TALE c d Zinc finger s RVD Bright field GFP Fokl 3 2 1 Fokl ZFN FP Control eG Fokl Fokl 1 2 3 500 bp DSB Correct repair NHEJ Deletion Wild type Insertion e Mutant WT b Deletions ITR Gact eGFP ITR * WT ZFN target eGFP-R Insertions/ deletions * * eGFP-L Figure 1 | Disruption of an eGFP transgene in G. bimaculatus using designed ZFNs. (a) Illustration of the binding and function of ZFNs and TALENs. Following the introduction of double-stranded breaks (DSB) in the DNA between targeted binding sites, these breaks are either repaired and digested again, or NHEJ can occur and deletions or insertions can result. In the latter case, gene function of the targeted gene is disrupted, and subsequent binding of ZFNs or TALENs does not result in FokI dimerization or the generation of DSBs. (b) Structure of the pBGact-eGFP transgene that is under the transcriptional control of the Gryllus cytoplasmic actin promoter (Gact) and flanked by ′5 and 3′ inverted terminal repeats (ITRs). The 9-bp binding sites for the ZFNs, eGFP-R and eGFP-L are in the boxes shown. (c) Detection of induced mutations into genomic DNA cleaved by Surveyor nuclease that was collected from embryos injected with both eGFP-R and eGFP-L mRNAs or eGFP-R mRNA alone (control) at 7 days postinjection. A product cleaved by Surveyor nuclease was detected in the genomic DNA from embryos injected with eGFP-R/L mRNAs (arrowhead). (d) Bright field (left panel) and fluorescence (right panel) phenotypes of 1G crickets injected with eGFP ZFN mRNA at 2 days after egg laying. While the embryos appeared to develop normally, some embryos did not exhibit eGFP fluorescence (indicated with arrows). Scale bar, 2.5 mm. (e) Sequence analysis of eGFP mutant alleles induced by eGFP ZFN mRNAs. Wild-type (WT) sequences are shown above the mutant sequences containing deletions (indicated with dashes) and/or insertions (shown in red letters). Asterisks indicate knockout mutations that included frame-shift and stop codon insertions (underlined). NatUre cOMMUNicatiONS | 3:1017 | DOI: 10.1038/ncomms2020 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited. All rights reserved. NatURE COMMUNicatiONS | DOI: 10.1038/ncomms2020 ARTICLE Here we report the generation of homozygous knockouts of an the mutations present resulted from NHEJ. Thus, a lack of eGFP- endogenous gene in G. bimaculatus via ZFN- and TALEN-based encoded fluorescence and mutations of the wild-type eGFP DNA targeted mutagenesis. A simple, efficient and broadly applicable sequence indicated that knockout of the eGFP transgene had been approach is described for the construction of gene knockouts, and achieved. In combination, these results show that engineered ZFNs this approach involves the direct injection of mRNAs encoding can induce mutations in a targeted locus of the cricket genome, and ZFNs or TALENs into embryos, combined with mutant detection this DSB induces error-prone NHEJ repair. assays that use the Surveyor (Cel-I) nuclease. Mutagenesis of an
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