Efficient CRISPR-mediated gene targeting and transgene replacement in the beetleTribolium castaneum

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.

Evolution of DNA Methylation across Insects

“…DNA methylation is found in all insect orders except Diptera, but the level of DNA methylation is fluid and phylogeny does not predict the pattern. Furthermore, the gain, loss, and duplication of DNMT1 suggests that there may be novel functions in different species, and justifies further studies using RNAi or CRISPR/Cas9 to mutate DNA methyltransferases (Gilles et al 2015). Finally, we found no association between level or presence of DNA methylation and the presence of sociality.…”

Ancient default activators of terminal photoreceptor differentiation in the pancrustacean compound eye: the homeodomain transcription factors Otd and Pph13

“…The RNAi knockdown approach, which has been highly efficient for characterizing gene functions [60], has recently been complemented by CRISPR-mediated gene knockout protocols in Tribolium and Daphnia [61,62]. This expanded range of genetic interference approaches can be expected to expedite the functional analysis of additional regulators in these models of pancrustacean compound eye development.…”

The Future of Cell Biology: Emerging Model Organisms

“…Moreover, CRISPR-based gene editing by homologous recombination allows for targeted changes to protein domains, meaning that the protein functions of emerging model organisms can be interrogated with the precision typically reserved for traditional models like worms and yeast. CRISPR methods may be a challenge for systems in which transgene expression has not yet been established, but DNA-free gene disruption using preassembled Cas9 protein and guide RNA gives hope, and has been shown to work in diverse systems including crustaceans, beetles, and even lettuce (Woo et al, 2015; Gilles et al, 2015; Martin et al, 2016). …”