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

Abstract

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, not related to vision, is desirable.

Evolutionary changes in germ granule mRNA content are driven by multiple mechanisms inDrosophila

“…Probe designs for nos were based on the coding sequence for all species, FBgn0002962 for D. mel (FlyBase), UniProt entry A0A6I8V5B3 for D. pse , and Q24710 for D. vir . For D. neb , the nos and pgc sequences were identified using the D. neb annotated long-read sequenced genome [46]. For D. mel, cycB , and gcl probes targeting their coding sequence were generated based on FlyBase sequences FBgn0000405, labeled with CAL Fluor Red 590 dye, and sequence FBgn0005695, labeled with CAL Fluor Red 610 Dye, respectively.…”

“…For osk sequences, UniProt entry numbers B4LXK5 and A0A6I8URE4 were used for D. vir and D. pse . For D. neb , rpl7 and osk sequences were determined using the D. neb annotated long-read sequenced genome [46]. For all expression assays, the presented fold gene expression levels are relative to D. mel and were calculated using the 2(-Delta Delta C(T)) method [49] using rpl7 as an internal control [20].…”

“…The D. mel neb nos 3′ UTR and D. mel pse nos 3′ UTR transgenes were created by first isolating species specific nos 3′ UTR from genomic DNA using PCR with primers with EcoRI and XhoI cut sites engineered at the ends of the forward and reverse primers and amplified with Phusion DNA Polymerase (NEB). Primers were designed based on the D. pse nos sequence used to designed the smFISH probe and the nos sequence for D. neb was identified from a long-read sequenced genome [46]. Next, we removed the nos 3′ UTR from a 4.3-kb D. mel genomic nos rescue fragment (a gift from the Gavis Lab [60]), using EcoRI and XhoI (NEB) and ligated species specific 3′ UTR with T4 DNA ligase (NEB).…”