RESEARCH REPORT TECHNIQUES AND RESOURCES Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum Anna F. Gilles 1,2, *, Johannes B. Schinko 1 and Michalis Averof 1,3, * ABSTRACT Gene-editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here, we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knockout and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knockouts; 71-100% carry such mutations in their germ line and transmit them to the next generation. We show that CRISPR-mediated gene knockout of the Tribolium E-cadherin gene causes defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marker transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs. KEY WORDS: Gene editing, Insect, Evo-devo INTRODUCTION Until recently, gene targeting was a privilege of the few; it was possible only in a small number of organisms and involved sophisticated, labor-intensive techniques. Gene targeting in mammals required first modifying an allele in embryonic stem cells, then selecting the few cells carrying the targeting event and transplanting them into blastocysts to generate chimeras in which these cells would hopefully populate the germ line and contribute to the next generation (Capecchi, 2005; Smithies et al., 1985; Thomas et al., 1986). In Drosophila, after several failed efforts, efficient gene targeting was developed using a method that required bringing together three different transgenes (Rong and Golic, 2000). These techniques were not applicable to most other organisms, in which cultured pluripotent cells and sophisticated genetics were unavailable. The invention of zinc-finger and transcription activator-like effector (TALE) nucleases marked a big step in our ability to target genes efficiently, by directing double-strand breaks to chosen sites in the genome and exploiting the cells’ endogenous DNA repair mechanisms to introduce changes at these sites (Carroll, 2014; Kim et al., 1996). Double-strand breaks are most frequently corrected by non-homologous end-joining (NHEJ) repair mechanisms, which can introduce small insertions or deletions (indels) at the site of repair. Less frequently, breaks are repaired through copying of a template that bears homologous sequences; such homology- directed repair (HDR) provides an opportunity to introduce specific changes into the locus via an engineered template. Zinc- finger and TALE nucleases proved to be extremely efficient for generating knockout and knock-in alleles compared with conventional gene targeting approaches. Their widespread use was limited mostly by the effort (and cost) required to customize their targeting specificity. The recent discovery of CRISPR/Cas nucleases, whose sequence specificity is guided by simple base complementarity between the target DNA and a small guide RNA (gRNA), provided a simple, efficient and affordable way of customizing nuclease specificity (Jinek et al., 2012; Sander and Joung, 2014). CRISPR/Cas nucleases consist of protein and RNA. Their specificity is determined by base complementarity with the 5′ end of the gRNA followed by the presence of a ‘protospacer adjacent motif’ (PAM) in the target sequence. In the most commonly used CRISPR system, derived from Streptococcus pyogenes, the PAM sequence is NGG. Thus, by cloning the appropriate targeting sequence (N 17-20 ) at the 5′ end of a gRNA, it is possible to generate nucleases targeting any sequence that conforms to N 17-20 NGG (where N can be any nucleotide). Owing to this straightforward way of generating nucleases with a chosen sequence specificity and to its high targeting efficiency in a range of organisms, CRISPR technology holds great promise for emerging model organisms (Gilles and Averof, 2014). In principle, CRISPR-mediated gene targeting should be applicable to all organisms. In practice, the effectiveness of this approach is constrained by our ability to deliver CRISPR/Cas nucleases to cells of interest (e.g. to the germ line), by the nature and efficiency of the organism’s DNA repair mechanisms and by our ability to identify and maintain the resulting mutants. These parameters will ultimately determine the feasibility and efficiency of gene targeting in a given species. Here, we present our effort to apply CRISPR technology in the beetle Tribolium castaneum and to establish methods and tools for efficient gene targeting in this species. Apart from being an important pest, infesting stored grain and grain products, Tribolium castaneum has emerged as an attractive experimental model for comparative developmental biology. Starting with classic genetic Received 7 April 2015; Accepted 29 June 2015 1 Institut de Gé nomique Fonctionnelle de Lyon (IGFL), École Normale Supé rieure de Lyon, 46 Allé ed’Italie, Lyon 69264, France. 2 École doctorale BMIC, Université Claude Bernard, Lyon 1, France. 3 Centre National de la Recherche Scientifique (CNRS), France. *Authors for correspondence (michalis.averof@ens-lyon.fr; anna.gilles@ens-lyon.fr) 2832 © 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 2832-2839 doi:10.1242/dev.125054 DEVELOPMENT