Modern molecular genetics studies necessitate the manipulation of genes in their endogenous
locus, but most of the current methodologies require an inefficient donor-dependent homologous
recombination step to locally modify the genome. Here we describe a methodology to efficiently generate
Drosophila knock-in alleles by capitalizing on the availability of numerous genomic MiMIC transposon
insertions carrying recombinogenic attP sites. Our methodology entails the efficient PhiC31-mediated integration
of a recombination cassette flanked by unique I-SceI and/or I-CreI restriction enzyme sites into an
attP-site. These restriction enzyme sites allow for double-strand break2mediated removal of unwanted
flanking transposon sequences, while leaving the desired genomic modifications or recombination cassettes.
As a proof-of-principle, we mutated LRRK, tau, and sky by using different MiMIC elements. We
replaced 6 kb of genomic DNA encompassing the tau locus and 35 kb encompassing the sky locus with
a recombination cassette that permits easy integration of DNA at these loci and we also generated a functional
LRRKHA knock in allele. Given that ~92% of the Drosophila genes are located within the vicinity (,35
kb) of a MiMIC element, our methodology enables the efficient manipulation of nearly every locus in the
fruit fly genome without the need for inefficient donor-dependent homologous recombination events.