Precise CRISPR-Cas9-mediated mutation of a membrane trafficking domain in the Drosophila vesicular monoamine transporter gene

James D Asuncion; Aditya Eamani; Ethan W Rohrbach; Elizabeth M Knapp; Sonali A Deshpande; Shivan L Bonanno; Jeremy E Murphy; Hakeem O Lawal; David E Krantz

doi:10.1016/j.crphys.2023.100101

Precise CRISPR-Cas9-mediated mutation of a membrane trafficking domain in the Drosophila vesicular monoamine transporter gene

Curr Res Physiol. 2023 Jun 20:6:100101. doi: 10.1016/j.crphys.2023.100101. eCollection 2023.

Authors

Affiliations

¹ Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
² Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
³ UCLA Neuroscience Interdepartmental Program, University of California, Los Angeles, CA, 90095, USA.
⁴ Department of Biological Sciences, Delaware State University, Dover, DE, USA, 19901, USA.

Abstract

Monoamine neurotransmitters such as noradrenalin are released from both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), the latter mediating extrasynaptic signaling. The contribution of synaptic versus extrasynaptic signaling to circuit function and behavior remains poorly understood. To address this question, we have previously used transgenes encoding a mutation in the Drosophila Vesicular Monoamine Transporter (dVMAT) that shifts amine release from SVs to LDCVs. To circumvent the use of transgenes with non-endogenous patterns of expression, we have now used CRISPR-Cas9 to generate a trafficking mutant in the endogenous dVMAT gene. To minimize disruption of the dVMAT coding sequence and a nearby RNA splice site, we precisely introduced a point mutation using single-stranded oligonucleotide repair. A predicted decrease in fertility was used as a phenotypic screen to identify founders in lieu of a visible marker. Phenotypic analysis revealed a defect in the ovulation of mature follicles and egg retention in the ovaries. We did not detect defects in the contraction of lateral oviducts following optogenetic stimulation of octopaminergic neurons. Our findings suggest that release of mature eggs from the ovary is disrupted by changing the balance of VMAT trafficking between SVs and LDCVs. Further experiments using this model will help determine the mechanisms that sensitize specific circuits to changes in synaptic versus extrasynaptic signaling.