SNP editing with CRISPR/Cas9

Single guide RNAs (sgRNAs) that bind near rs1663689 were designed with an online tool (http://crispr.mit.edu/) and cloned into pX458, which expressed green fluorescent protein and Cas9 (Addgene, pSpCas9(BB)‐2A‐GFP, Plasmid 48138). A 68 bp double‐stranded DNA (dsDNA) containing “T” or “C” at rs1663689 was used for homology‐directed repair. A total of 4 μg pX458 containing sgRNAs and 14 pmol of dsDNA donor were cotransfected into A549 and H460 cells in 60 mm culture dishes following the instructions of the Lipofectamine™ 3000 Reagent Protocol (1684916, Thermo Fisher). Thirty‐six hours after transfection, FACS sorting was carried out using the expression of GFP as a selection marker. Single cells were cultured in 96‐well plates to form clones. Genomic DNA was extracted using a Genomic DNA Purification Kit (EE101‐02, TransGen) according to the manufacturer's instructions. The rs1663689 region was amplified by PCR and verified by Sanger sequencing. Primers were listed in Table EV2.