CRISPR/Cas9 and FLP-FRT mediated multi-modular engineering of the cis-regulatory landscape of the bithorax complex of Drosophila melanogaster

Abstract

Abstract The Homeotic genes or Hox define the anterior-posterior (AP) body axis formation in bilaterians and are often present on the chromosome in an order which is collinear to their function across the AP axis. However, there are many cases wherein the Hox are not collinear, but their expression pattern is conserved across the AP axis. The expression pattern of Hoxis attributed to the cis-regulatory modules (CRMs) consisting of enhancers, initiators, or repressor elements that together regulate the genes in a segment-specific manner. In the Drosophila melanogaster Hoxcomplex, the bithorax complex (BX-C), even the CRMs are organized in an order that is collinear to their function in the thoracic and abdominal segments. In the present study, we performed in-silico analysis of the available ChIP data followed by systematic curation of experimentally validated regions of the BX-C gene, Abd-B, to generate functionally relevant map of the regulatory landscape. Next, the regulatorily inert regions were targeted using CRISPR/Cas9 to generate a series of transgenic lines with the insertion of FRT sequences. Further, these FRT lines are repurposed to shuffle the CRMs associated with Abd-B to generate modular deletion, duplication, or inversion of multiple CRMs. The rearrangements yielded entirely novel phenotypes in the fly suggesting the requirement of such complex manipulations to address the significance of higher order arrangement of the CRMs. The functional map and the transgenic flies generated in this study are important resource to decipher the collective ability of multiple regulatory elements in eukaryotic genome to function as complex modules.