CRISPR sgRNA Vector Construction

We replaced the unc-119 target sequence in pU6::unc-119 sgRNA vector (Friedland et al. 2013 (link)) with the desired target sequence using overlap extension PCR. The pU6::unc-119 sgRNA vector was diluted to 2 ng/µl and used as a template to generate two overlapping fragments. The first was amplified using the primers CMo16428 and sgRNA R, resulting in the U6 promoter fused to the GN₁₉ target sequence (U6p::GN₁₉). The second was amplified using the primers CMo16429 and sgRNA F, resulting in the GN₁₉ target sequence fused to the sgRNA scaffold and U6 3′-UTR. These two PCR products were mixed together, diluted 1:50, and used as a template for a PCR reaction with primers CMo16428 and CMo16429. The resulting pU6::target sequence::sgRNA scaffold::U6 3′-UTR fusion products were gel purified and inserted into the pCR-Blunt II-TOPO vector (Invitrogen, no. K2800-20). We used iProof high-fidelity DNA polymerase (Bio-Rad, no. 172-5300) in all PCR reactions above to minimize errors of PCR amplification, and all the constructs were confirmed by DNA sequencing. Primers sequences are listed in Table S2.