The pl018 Drosophila expression vector (28) was digested with Bbs I (Thermo Fisher Scientific, no. ER1011) for 30 min at 37°C, and the linearized backbone vector was purified by polymerase chain reaction (PCR) purification. sgRNA sequences were designed using the Drosophila RNAi Screening Core sgRNA design tool (www.flyrnai.org/crispr2/). Sense and antisense vhl oligonucleotides containing appropriate overhangs for ligation into the Bbs I–digested vector were synthesized by Integrated DNA Technologies (IDT) (vhl sense, 5′-GTTCGTCTGTACTGGGTGTGCGAGC-3′; vhl antisense, 5′-AAACGCTCGCACACCCAGTACAGAC-3′).

An equimolar ratio of oligonucleotides (0.1 nmol of each sense and antisense oligonucleotide) was then annealed and phosphorylated by T4 Polynucleotide Kinase (New England Biolabs, no. M0201). Annealing and phosphorylation were carried out using a 30-min incubation at 37°C followed by a 5-min incubation at 95°C. The incubation temperature was then lowered by 5°C/min until a final temperature of 25°C was reached. The annealed phosphorylated oligonucleotides were then ligated into the Bbs I–digested pl018 by incubating for 5 min at room temperature with T7 Ligase (Enzymatics, no. L602L). A 2-μl aliquot of the ligation reaction was then transformed into chemically competent Escherichia coli cells. Plasmid DNA from ampicillin-resistant colonies was evaluated by high-resolution melt assay (HRMA) as previously described (21) and further confirmed by deep amplicon sequencing.

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