Scarless Modification of the Drosophila Genome Near Any Mapped attP Sites

Abstract

AbstractHere we describe a Drosophila genome engineering technique that can scarlessly modify genomic sequences near any mapped attP attachment site previously integrated by transposon mobilization or gene targeting. This technique combines two highly efficient and robust procedures: phiC31 integrase–mediated site‐specific integration and homing endonuclease–mediated resolution of local duplications. In this technique, a donor fragment containing the desired mutation(s) is first integrated into a selected attP site near the target locus by phiC31 integrase–mediated site‐specific integration, which creates local duplications consisting of the mutant‐containing donor fragment and the wild‐type target locus. Next, homing endonuclease–induced double‐stranded DNA breaks trigger recombination between the duplications and resolve the target locus to generate scarless mutant alleles. In every step, the desired flies can be easily identified by patterns of dominant markers, so no large‐scale screens are needed. This technique is highly efficient and can be used to generate scarless point mutations, insertions, and deletions. The availability of large libraries of mapped attP site–containing transposon/CRISPR insertions in Drosophila allows the modification of more than half of the euchromatic Drosophila genome at a high efficiency. As more and more attP‐containing insertions are generated and mapped, this technique will be able to modify larger portions of the Drosophila genome. The principles of this technique are applicable to other organisms where modifications to the genome are feasible. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Verifying attP‐containing insertionsSupport Protocol: Extracting genomic DNABasic Protocol 2: Generating the donor plasmidBasic Protocol 3: Injecting the donor plasmid and establishing transformant stocksBasic Protocol 4: Verifying the transformantsBasic Protocol 5: Generating the final scarless allelesBasic Protocol 6: Verifying the final alleles