CRISPR-Cas9 sgRNA Design and Lentiviral Production

Three sgRNA sequences targeting Napa (5′-CTTGAACATGTTCGCCGCTC-3′, 5′-AGATTGCTCATAGTGGGCGA-3′, 5′-TGCTGGGAACGCTTTCTGCC-3′) and Ppp2cb (5′-TCTCGCACAGCGTCCGCACT-3′, 5′-ATACGAACTACCTATTCATG-3′, 5′- CGCAATATTGTGATGCGCTC-3′) were designed using CHOPCHOP (version 3) [21 (link)]. Custom oligos (Integrated DNA Technologies, Coralville, IA, USA) were phosphorylated in vitro with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA), annealed by heating/cooling in a thermocycler, ligated into the BsmBI site of pSECC (Addgene # 60820) with T4 DNA Ligase (New England Biolabs, Ipswich, MA, USA), and plasmid was purified from Stbl3 E. coli (Thermofisher, Waltham, MA, USA). Lentiviral particles for each individual sgRNA construct and a scrambled control (5′-GCGAGGTATTCGGCTCCGCG-3′) [22 (link)] were generated by Lipofectamine Plus/LTX-mediated transfection of 293FT cells with pSECC, pCMV-VSV-G (Addgene #8454), pMDLg/pRRE (Addgene #12251), and pRSV-Rev (Addgene #12253). The supernatant containing viral particles was collected at 48 h post-transfection.