The authors have developed two conditional gene manipulation systems, cCCTomics and C-cCCTomics, to target all chemical transmission (CCT) genes in Drosophila.
cCCTomics utilizes GFP-RNAi and Flp-out strategies to achieve near-complete disruption of target CCT genes. This system leverages previously generated CCT knockin lines, where the GFP coding sequence is fused to the 3' end of each gene and the gene span is flanked by FRT sequences. Expressing GFP-RNAi or Flp recombinase can efficiently eliminate GFP signals, indicating effective gene disruption.
To further simplify the conditional manipulation of CCT genes, the authors developed C-cCCTomics, a CRISPR/Cas9-based system. They generated UAS-sgRNA transgenic lines targeting all 209 defined CCT genes and combined them with UAS-Cas9 variants. The authors found that Cas9 variants fused with chromatin-modulating peptides, Cas9.M6 and Cas9.M9, exhibited significantly higher gene disruption efficiency compared to the unmodified Cas9.
The authors then applied these conditional gene manipulation tools to dissect the chemoconnectome of Drosophila clock neurons. Intersecting Clk856-Gal4 with CCT gene knockin lines, they identified 43 CCT genes expressed in various subsets of clock neurons.
Further functional analysis using C-cCCTomics revealed that the neuropeptide CNMa and its receptor CNMaR play an antagonistic role with PDF-PDFR signaling in regulating morning anticipation behavior. Specifically, knockout of CNMa or CNMaR in clock neurons, particularly the PDF-PDFR co-expressing DN1p neurons, led to advanced morning activity.
These results demonstrate the effectiveness of the authors' conditional CCTomics toolkit and its application in dissecting neural circuits underlying complex behaviors.
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by Mao,R., Yu,J... 於 www.biorxiv.org 09-27-2023
https://www.biorxiv.org/content/10.1101/2023.09.26.559642v2深入探究