Nebulous without white: annotated long-read genome assembly and CRISPR/Cas9 genome engineering in Drosophila nebulosa

• Published in: G3 : genes - genomes - genetics Vol. 12; no. 11
• Main Authors: Sottolano, Christopher J, Revaitis, Nicole T, Geneva, Anthony J, Yakoby, Nir
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 04.11.2022
• Subjects: Animals; Animals, Genetically Modified; CRISPR-Cas Systems; Drosophila - genetics; Drosophila melanogaster - genetics; Gene Editing; Genome Report; Male; genome editing; genome sequencing; CRISPR/Cas9; PacBio
• ISSN: 2160-1836; 2160-1836
• DOI: 10.1093/g3journal/jkac231

The diversity among Drosophila species presents an opportunity to study the molecular mechanisms underlying the evolution of biological phenomena. A challenge to investigating these species is that, unlike the plethora of molecular and genetics tools available for D. melanogaster research, many other species do not have sequenced genomes; a requirement for employing these tools. Selecting transgenic flies through white (w) complementation has been commonly practiced in numerous Drosophila species. While tolerated, the disruption of w is associated with impaired vision, among other effects in D. melanogaster. The D. nebulosa fly has a unique mating behavior which requires vision, and is thus unable to successfully mate in dark conditions. Here, we hypothesized that the disruption of w will impede mating success. As a first step, using PacBio long-read sequencing, we assembled a high-quality annotated genome of D. nebulosa. Using these data, we employed CRISPR/Cas9 to successfully disrupt the w gene. As expected, D. nebulosa males null for w did not court females, unlike several other mutant strains of Drosophila species whose w gene has been disrupted. In the absence of mating, no females became homozygous null for w. We conclude that gene disruption via CRISPR/Cas9 genome engineering is a successful tool in D. nebulosa, and that the w gene is necessary for mating. Thus, an alternative selectable marker unrelated to vision is desirable.