Demonstration ofin vivoengineered tandem duplications of varying sizes using CRISPR and recombinases inDrosophila melanogaster

Abstract

AbstractTandem gene duplicates are important parts of eukaryotic genome structure, yet the phenotypic effects of new tandem duplications are not well-understood, in part owing to a lack of techniques to build and modify them. We introduce a method, Recombinase-Mediated Tandem Duplication (RMTD), to engineer specific tandem duplicationsin vivousing CRISPR and recombinases. We describe construction of four different tandem duplications of theAlcohol Dehydrogenase(Adh) gene inDrosophila melanogaster, with duplicated block sizes ranging from 4.2 kb to 20.7 kb. Flies with theAdhduplications show elevated ADH enzyme activity over unduplicated single copies. This approach to engineering duplications is combinatoric, opening the door to systematic study of the relationship between the structure of tandem duplications and their effects on expression.