Tag-RFP-T-SH3BP5 and SH3BP5L fusions were subcloned into the retroviral plasmid pMXs-IRES-Puro (Clontech) using the NotI/EcoRI restriction sites and InFusion HD cloning kit (Clontech).
sgRNA sequences targeting dog SH3BP5, SH3BP5L, TNKS, TNKS2, and RNF146 were selected from the CRISPOR design tool (http://www.crispor.tefor.net) by entering the cDNA sequence of specific exons (Haeussler et al., 2016 (link)). Guide sequences were cloned into the plasmid pLCKO using BfuAI and NsiI sites (Table 1).
Two shRNAs against dog Rab11a were obtained from the RNAi consortium. Scrambled control (shSCM) hairpin was cloned into pLKO.1 (Addgene #10878) by annealing oligos into pLKO.1 digested with EcoRI and AgeI (Table 2).