Investigating the use of enhancer trapping and Cas9-induced rearrangements to characterize cis-regulatory elements in the Drosophila gene hindsight

Abstract

The gene hindsight (hnt) encodes a transcription factor that is essential for all stages of Drosophila development. Expression of hnt has been observed to promote cell specialization and differentiation of several cell lineages at different stages. Consequently, the temporal and spatial expression of hnt is both dynamic and complex. A tremendous amount of research in this gene has been dedicated to its protein activity; however, little is known about the regulatory mechanisms that modulate its expression. Two genetic approaches have been applied to the 5’ region of hnt to investigate its role in hnt regulation, including enhancer-based techniques and Cas9 multiplexing. Additionally, the use of Cas9 multiplexing was investigated for its feasibility in inducing chromosomal rearrangements (CRs). This was performed in the upstream region of hnt, as a means to study potential regulatory sequences associated with the gene. In investigating this region with an enhancer trap-based method, putative elements that directly influence hnt expression have been characterized in terms of the cells and stages in which they influence hnt expression. In investigating the upstream region of hnt using Cas9- induced CRs, two lines with putative deletions in the ruby (rb) region have been recovered. Both lines display misexpression of hnt, indicating the presence of hnt-associated elements within the deleted region. While deletions using this system was successful, no translocations or inversions were recovered from Cas9 multiplexing. Results indicate that the use of Cas9 multiplexing without the use of homology-based techniques does not provide a feasible method for inducing CRs.