Generation of a crk null allele

The crk null allele, crk^ΔattP, was generated by using CRISPR to replace the entire crk locus (beginning 110 base pairs upstream of transcription start site and ending 99 base pairs beyond the 3′UTR) with a 50–base pair attP phage recombination site and positive marker (a loxP-flanked 3xP3-DsRed cassette) as described in Gratz et al. (2014) . Briefly, guide RNAs (gRNAs ) flanking the crk locus were selected using flyCRISPR Target Finder (http://tools.flycrispr.molbio.wisc.edu/targetFinder/) to identify 19- to 20–base pair CRISPR targets with NGG PAM sites and zero predicted off-target effects using maximum stringency. Annealed gRNA oligo nucleotides were cloned into the BbsI site of pU6-BbsI-chiRNA. We generated a dsDNA donor template for homology-directed repair by cloning upstream and downstream flanking regions of the crk locus into the AarI and SapI sites of pHD-DsRed-attP, respectively. An 815–base pair upstream homology region, extending from 5′-CTTGAGCATGCAAAGGAATG-… to …GCTGACAGTACGTCCT-3′ and flanked by AarI sites, was synthesized as a gBlocks Gene Fragment (IDT, Coralville, IA). A 1-kb downstream homology region was PCR-amplified from genomic DNA using the primers described below. An injection mixture containing both U6-gRNA plasmids (75 ng/µl each) and the dsDNA template (250 ng/µl) was injected into y¹, M{vas-Cas9.RFP-}ZH-2A, w¹¹¹⁸ flies. Injections were performed by BestGene (Chino Hills, CA). Surviving G₀ animals were outcrossed to y, w and CRISPR-edited lines were identified by the presence of DsRed eye fluorescence. DsRed-positive G₁ animals were further outcrossed to y, w to remove M{vas-Cas9.RFP-}ZH-2A and subsequently established as stocks over In(4) ci^D, ci^D, pan^ciD. Deletion of the crk locus was verified by PCR using the primers described below.

Oligo nucleotides for gRNA1:

Target (PAM underlined): GCTGACAGTACGTCCTAAAGGG

Sense oligo: 5′-CTTCGCTGACAGTACGTCCTAAA-3′

Antisense oligo: 5′-AAACTTTAGGACGTACTGTCAGC -3′

Oligo nucleotides for gRNA2:

Target (PAM underlined): GAATTGTGTAACTAAACGGTAGG

Sense oligo: 5′-CTTCGAATTGTGTAACTAAACGGT-3′

Antisense oligo: 5′-AAACACCGTTTAGTTACACAATTC-3′

Primers for PCR amplification of the downstream homology region (Sap1 sites are in bold; target sequences are underlined):

Forward: 5′-GACTGCTCTTCATATTGCTACAGAAAACCCTT­TGG -3′

Reverse: 5′-GACTGCTCTTCAGACGAACATAGACAGCACAA­GTTTGTTG -3′

Primers for PCR verification of crk locus deletion:

Detection of DsRed:

Forward: 5′-CGAGGACGTCATCAAGGAGT-3′

Reverse: 5′-GGTGATGTCCAGCTTGGAGT-3′

Flanking upstream HR:

Forward: 5′-AATTGCGCGCTGTTAAAAAT-3′

Reverse: 5′-GCTTCGAGCCGATTGTTTAG-3′

Flanking downstream HR:

Forward: 5′-GTGGACTCCAAGCTGGACAT-3′

Reverse: 5′-TAACAGGCAAAAACGCTTCC-3′