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

AbstractThe 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. The expression pattern of Hox genes is attributed to the cis-regulatory modules (CRMs) that regulate the genes in a segment-specific manner. In the bithorax complex (BX-C), one of the two Hox complexes in Drosophila melanogaster, even the CRMs consisting of enhancers, initiators, insulators, and Polycomb/trithorax response elements are organized in order that is collinear to their function in the thoracic and abdominal region. Much of these findings are based on the analysis of hundreds of mutations in the BX-C. However, targeted genomic rearrangements comprising of duplications, inversions, etc., that can reveal the basis of collinearity and the number of regulatory modules with respect to body segments have not been reported. In the present study, we generated a series of transgenic lines with the insertion of FRT near the regulatory domain boundaries, to shuffle the CRMs associated with the posterior Hox, Abd-B, of the BX-C. Using these FRT lines, we created several alterations such as deletion, duplication, or inversion of multiple CRMs to comprehend their peculiar genomic arrangement and numbers in the BX-C.