CRISPR- Mediated Deletions of the knirps Cis-Regulatory Module in Drosophila

Wing-vein patterning in Drosophila is a well-understood developmental system that serves as a paradigm for studying the link between gene expression and tissue morphology. Deciphering the transcriptional information contained within cis-regulatory modules (CRMs) is integral to understanding such a relationship. I focused my work on an enhancer element within the knirps locus which had been previously identified as an essential component responsible for the development of the L2 wing vein. Previous studies also characterized regulatory alleles of knirps, named radius incompletus, which consisted of deletions and substitutions within the enhancer that greatly reduce or eliminate knirps expression and produce a variety of loss-of-vein phenotypes. The aim of my project is to perform an in vivo sequential analysis of the function of the knirps enhancer. Using the CRISPR/Cas9 genome editing system, I was able to generate sequential deletions of conserved regions within the knirps locus and observe their direct output in the wing. From the mutants recovered, I was able to reproduce phenotypes typical of classic ri alleles of differing severity and compared the vein deletion patterns to the sequences of their altered enhancers. The mutants generated from this study provide insight on the functionality of enhancers, allowing for an in-depth analysis of the module's transcriptional activity as a unit in the context of its native genom