Scarless engineering of theDrosophilagenome near any site-specific integration site

Abstract

We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This 2-step method combines phiC31 integrase mediated site-specific integration and homing nuclease mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.

Transcription factor paralogs orchestrate alternative gene regulatory networks by context-dependent cooperation with multiple cofactors

“…1e), allowing us to use the same anti-FLAG antibody to obtain genome-wide binding data for both Hox paralogs (see Methods, Extended Data Fig. 2 and ref 13 ). Multiple verified alleles for both genotypes ( 3xFLAG-Scr and 3xFLAG-Ubx ) were homozygous viable and fertile, and did not show any noticeable developmental delays or defects.…”

“… e. Schematics of the 3xFLAG tagged Scr (see Methods) and Ubx alleles generated by genome targeting 13 . The wide boxes (orange for Scr and green for Ubx ) indicate coding regions, and the homeobox is colored purple.…”

“…All final stocks were also screened under fluorescent scope to make sure there was no eye-specific RFP expression, and were verified by southern blot analysis and DNA sequencing. The generation of the 3xFLAG-Ubx allele was described in 13 . 3xFLAG-Scr C 18–6 , 3xFLAG-Ubx 7 and 3xFLAG-Scr(YPWM-AAAA) D8-16 alleles were used throughout this study.…”

SpyChIP identifies cell type-specific transcription factor occupancy from complex tissues

“…The scarless SpyTag-Ubx allele was generated using a method we previously described (7). Briefly, a fragment of Ubx genomic DNA containing the SpyTag inserted at the N-terminal end of the Ubx ORF was integrated into the endogenous Ubx locus by phiC31 integrase mediated site-specific recombination.…”

“…Double-stranded DNA breaks were then introduced to stimulate homologous recombination and repair the endogenous Ubx to the final scarless SpyTag-Ubx allele. The landing site for site-specific recombination in the Ubx locus has been described in detail, and the donor plasmid was generated similarly as before (7). The SpyTag sequence was inserted by overlapping extension PCR.…”