Lenticrisprv2 blast plasmid

Human TRIT1 mutant cells and control cells were generated using the CRISPR/Cas9 system essentially as described previously (Shalem et al. 2014 (link)). Briefly, sense and antisense oligonucleotides encoding a single guide RNA (sgRNA) against TRIT1 gene or a control sgRNA (not targeting human genome) (Supplemental Table 1; Shalem et al. 2014 (link)) were cloned into the BsmBI sites of lentiCRISPR v2 Blast plasmid (Addgene #83480). Lentiviruses were generated in HEK293FT cells by transfecting the sgRNA sequence-containing lentiCRISPR v2 Blast plasmid, psPAX2 plasmid (Addgene #12260) and pMD2.G plasmid (Addgene #12259) with Lipofectamine 3000 (Invitrogen). Fresh HEK293FT cells were transduced with the generated viruses, followed by blasticidin selection of the transduced cells. Subsequently, single clones were acquired by diluting the cells in 96-well plates followed by expansion of the clones. The target region of the genome in each clone was PCR-amplified and sequenced with conventional Sanger sequencing, using the primers listed in Supplemental Table 1.