sgRNA expression vectors: for expression of single sgRNAs, oligonucleotide pairs (Supplementary Table 3 ) were annealed and cloned into BbsI-digested pCFD3-dU6-3gRNA (Addgene #49410), as described44 . To express multiple sgRNAs from the same vector backbone, oligonucleotide pairs (Supplementary Table 4 ) were used for PCR and inserted into pCFD5 (Addgene #73914) via Gibson Assembly, as described45 (link).
Donor vectors for homologous recombination: to generate an eGFP-expressing donor vector (pHD-Stinger-attP), the fluorophore was excised from pStinger46 (link) with NcoI/HpaI and used to replace the DsRed sequence in NcoI/HpaI-digested pHD-DsRed-attP (Addgene plasmid #51019)47 (link). Homology arms (1-1.6 kb) for individual target genes were amplified from D. sechellia (Drosophila Species Stock Center [DSSC] 14021-0248.07), D. simulans (DSSC 14021-0251.195) or D. melanogaster (Research Resource Identifier Database:Bloomington Drosophila Stock Center [RRID:BDSC]_58492) genomic DNA and inserted either into pHD-DsRed-attP or pHD-Stinger-attP via restriction cloning. Details and oligonucleotide sequences are available from the corresponding authors upon request.
Transgenic source of Cas9: pBac(nos-Cas9,3XP3-YFP) (gift of D. Stern) was integrated into D. sechellia (DSSC 14021-0248.07) via piggyBac transgenesis. The insertion was mapped to the fourth chromosome using TagMap48 (link).
Donor vectors for homologous recombination: to generate an eGFP-expressing donor vector (pHD-Stinger-attP), the fluorophore was excised from pStinger46 (link) with NcoI/HpaI and used to replace the DsRed sequence in NcoI/HpaI-digested pHD-DsRed-attP (Addgene plasmid #51019)47 (link). Homology arms (1-1.6 kb) for individual target genes were amplified from D. sechellia (Drosophila Species Stock Center [DSSC] 14021-0248.07), D. simulans (DSSC 14021-0251.195) or D. melanogaster (Research Resource Identifier Database:Bloomington Drosophila Stock Center [RRID:BDSC]_58492) genomic DNA and inserted either into pHD-DsRed-attP or pHD-Stinger-attP via restriction cloning. Details and oligonucleotide sequences are available from the corresponding authors upon request.
Transgenic source of Cas9: pBac(nos-Cas9,3XP3-YFP) (gift of D. Stern) was integrated into D. sechellia (DSSC 14021-0248.07) via piggyBac transgenesis. The insertion was mapped to the fourth chromosome using TagMap48 (link).
