Elsevier

Methods

Volume 69, Issue 1, 15 August 2014, Pages 17-21
Methods

Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs

https://doi.org/10.1016/j.ymeth.2014.05.006Get rights and content

Abstract

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms.

Introduction

The two-spotted cricket, Gryllus bimaculatus (Orthoptera: Gryllidae), is one of the most abundant cricket species. G. bimaculatus inhabits tropical and subtropical regions of Asia, Africa, and Europe. It can be easily bred in the laboratory and has been widely used to study insect physiology and neurobiology [1]. This species was recently established as a model system for studies on molecular mechanisms of development and regeneration. To analyze gene functions in G. bimaculatus, the RNA interference (RNAi) technique can be used. For genes involved in embryonic development, double-stranded RNA (dsRNA) is injected into the body cavity of adult female crickets [2]. RNAi effects are observed through the subsequently laid eggs. To study regeneration mechanisms of the leg, dsRNA can be injected into the body cavity of the nymph [3], followed by amputation of the metathoracic tibia and observation of the RNAi effects during regeneration of the lost part of the leg.

We recently established a technique to generate transgenic crickets with the piggyBac transposase [4], [5]. Using eGFP-expressing transgenic lines, we performed live imaging analysis of fluorescently labeled embryonic cells and nuclei. Blastoderm cells were found to move dynamically, retaining their positional information to form the posteriorly localized germ anlage [4]. The generation of transgenic crickets would also be useful for analyzing the functions of genes and cis-regulatory elements.

Although the above RNAi and transgenic systems are effective for analyzing gene function, each system has some shortcomings. For example, gene functions can be easily and efficiently inhibited by RNAi, but they cannot be inhibited completely because some RNA can remain un-degraded. In the case of piggyBac-based transgenesis, we cannot control both the copy number and the genomic locus of a transgene because, by nature, transgenes are randomly integrated into a genome. Thus, a technique is needed for modifying the cricket genome at a specific site, in order to conduct further sophisticated gene-functional analyses.

Transcription activator-like (TAL) effector nucleases (TALENs) can be used to induce targeted DNA double-strand breaks (DSBs) into specific regions of the genome [6]. TALENs consist of an engineered array of TAL effector repeats fused to the FokI cleavage domain. DNA-binding domains of TALENs have tandem repeat units of 33–35 amino acids in length. The nucleotide binding preference is determined by two adjacent amino acids, known as the repeat variable di-residue [7]. FokI requires dimerization to cleave DNA. Dimerization is achieved through the binding of two TALEN molecules to contiguous target sequences in each DNA strand, separated by a spacer sequence. Induction of DSBs by TALENs activates the DNA damage response [8]. A DSB can be repaired by nonhomologous end joining, in which short insertions or deletions are generated at the cleavage site [9], or by homologous recombination with a DNA template, in which gene knock-in results in a perfect repair or a sequence replacement (if a modified template is used) [10].

TALEN-mediated mutations have been induced in several organisms, including fruit flies [11], zebrafish [12], rats [13], and mice [14]. In these animals, both somatic and germline mutations were shown [11], [12], [13], [14]. However, obtaining constitutional homozygous mutants is laborious in cases without specific genetic markers to breed with, or without morphologically detectable phenotypes to identify individuals with a mutated allele.

To facilitate the production of homozygous knockout animals via genome editing, we examined an effective strategy for selecting animals with a mutant allele, independent of their genetic tractability or their phenotypic characteristics. We reported the first research in hemimetabolous insects to show the effectiveness of TALENs for generating knockout animals [15]. We designed TALENs targeted to the G. bimaculatus laccase2 (Gblac2) locus. This gene was selected based on previous studies, in which RNAi targeting of Gblac2 in the nymphal stages of the cricket resulted in loss of cuticle tanning after molting.

After in vitro transcription, TALEN mRNAs were injected into cricket eggs just after fertilization. Approximately 17% of the fifth instar G0 larvae generated were somatic mutants with a mosaic cuticle pattern, with or without tanning. To generate knockout crickets, we designed a two-step screening method based on mutant detection assays using SURVEYOR nuclease. Mutations transmitted from the founder crickets were detected in ∼17% of G0 adults in the first round of screening. Heterozygous offspring were selected with mutation detection assays in the second round of screening. Subsequent sibling crosses created homozygous knockout crickets exhibiting the cuticle tanning defect phenotype (Fig. 1).

In this article, we provide protocols for generating homozygous knockouts of an endogenous gene in G. bimaculatus via TALEN-based targeted mutagenesis. This approach can be used to generate knockout animals, independent of the specific genotype or phenotype. In principle, these protocols can be adapted to any organism for which TALEN mRNA can be injected into eggs and the organism reared through a few generations.

Section snippets

Breeding crickets

  • 1.

    TetraFin (Tetra) for adult food.

  • 2.

    TetraGold (Tetra) for nymph food.

  • 3.

    Paper towels for dishes to collect eggs and for water tubes.

  • 4.

    Insect cages.

  • 5.

    Petri dishes.

  • 6.

    50-mL plastic centrifuge tubes.

Injection

  • 1.

    Glass capillary (G-1, Narishige).

  • 2.

    Micropipette puller (P-1000, Sutter Instruments).

  • 3.

    Glass slide (S244, Matsunami).

  • 4.

    Double-sided tape (TW-185D, Nichiban).

  • 5.

    Inverted microscope, 10X eyepiece lens and 10X objective lens (DM IRB, Leica) (Fig. 2A).

  • 6.

    50-mL glass syringe (TOP) (Fig. 2A).

  • 7.

    Micromanipulator (Leitz Micromanipulator M)

Maintenance of wild-type crickets

Rear nymphs and adults at 29 °C and 50% humidity under a 10-h light: 14-h dark photoperiod. Feed nymphs and adults with artificial fish food (e.g., TetraFin for adults and TetraGold for nymphs). Under these conditions, first to third instar nymphs will molt every 2 or 3 days, fourth to sixth instar nymphs will molt every 4 or 5 days, seventh and eighth instar nymphs will molt every week, and adults will emerge after the eighth molt. The generation time of the cricket is about 2 months.

Maintain

Acknowledgment

This work was supported by JSPS KAKENHI (23687033/25650080) to T.M., (22124003) to T.M. and S.N.

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