CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous <i>Drosophila kni</i>-L2 CRM reveals unexpected complexity.

Abstract

The <i>knirps</i> (<i>kni</i>) locus encodes transcription factors required for induction of the L2 wing vein in <i>Drosophila</i>. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for <i>kni</i> expression in the L2 vein primordium. Phenotypic analysis of these '<i>in locus</i>' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis.