Clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated mutagenesis and phenotype rescue by piggyBac transgenesis in a nonmodel Drosophila species

• Published in: Insect molecular biology Vol. 25; no. 4; pp. 355 - 361
• Main Authors: Tanaka, R., Murakami, H., Ote, M., Yamamoto, D.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.08.2016
• Subjects: Animals; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; Base Sequence; Clustered Regularly Interspaced Short Palindromic Repeats; courtship; Drosophila - classification; Drosophila - genetics; Drosophila - metabolism; Drosophila melanogaster; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Drosophila subobscura; Eye Proteins - genetics; Eye Proteins - metabolism; Female; Gene Transfer Techniques; genes; genetic techniques and protocols; genome editing; germ cells; germline transformation; Male; Mutagenesis; mutants; Mutation; Phenotype; phylogeny; pigmentation mutations; Species diversity; transgenesis; visible marker; genome editing; visible marker; germline transformation; pigmentation mutations
• ISSN: 0962-1075; 1365-2583; 1365-2583
• DOI: 10.1111/imb.12232

How behavioural diversity emerged in evolution is an unexplored subject in biology. To tackle this problem, genes and circuits for a behaviour need to be determined in different species for phylogenetic comparisons. The recently developed clustered regulatory interspaced short palindromic repeats/CRISPR associated protein9 (CRISPR/Cas9) system made such a challenge possible by providing the means to induce mutations in a gene of interest in any organism. Aiming at elucidating diversification in genetic and neural networks for courtship behaviour, we attempted to generate a genetic tool kit in Drosophila subobscura, a nonmodel species distantly related to the genetic model Drosophila melanogaster. Here we report the generation of yellow (y) and white mutations with the aid of the CRISPR/Cas9 system, and the rescue of the y mutant phenotype by germline transformation of the newly established y mutant fly line with a y+‐marked piggyBac vector. This successful mutagenesis and transformation in D. subobscura open up an avenue for comprehensive genetic analyses of higher functions in this and other nonmodel Drosophila species, representing a key step toward systematic comparisons of genes and circuitries underlying behaviour amongst species.