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

Elife. 2017 Dec 23;6:e30281. doi: 10.7554/eLife.30281.

Abstract

The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. 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 kni expression in the L2 vein primordium. Phenotypic analysis of these 'in locus' 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.

Keywords: CRISPR; D. melanogaster; Drosophila; active genetics; developmental biology; evolutionary biology; genomics; in-locus; knirps; stem cells; wing vein.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Drosophila Proteins / genetics*
  • Drosophila melanogaster / classification
  • Drosophila melanogaster / genetics*
  • Drosophila melanogaster / growth & development
  • Gene Expression Regulation, Developmental*
  • Regulatory Sequences, Nucleic Acid*
  • Repressor Proteins / genetics*
  • Species Specificity
  • Wings, Animal / growth & development*
  • Wings, Animal / physiology

Substances

  • Drosophila Proteins
  • Repressor Proteins
  • kni protein, Drosophila