2.12. Establishment of CRISPR/Cas9 based specific gene knockout in A549 cells

sgRNA sequences targeting exons of the human STAT3 were designed by using the Millipore&Sigma CRISPR design tool (https://www.milliporesigmabioinfo.com/bioinfo_tools/faces/informatics.xhtml). The predesigned sequence with the fewest predicted off-target cleavage sites was selected. Oligos were synthesized, annealed, and ligated to a FastDigest BsmBI (Thermo, FD0454) digested lentiCRISPR v2 plasmid by using T4 DNA ligase (Thermo, EL0014). LentiCRISPR v2 plasmid containing sgRNA with no human genome target sequence (NT) was used as a control.

For lentivirus package, HEK 293T cells (1.5 × 10⁶) were transfected with STAT3-sgRNA LentiCRISPR v2 plasmid (800 ng) or NT-sgRNA LentiCRISPR v2 plasmid (800 ng) and two packaging plasmids (pCMV-dR8.91 (1200 ng) coding HIV-Gag and HIV-Pol proteins and pMD2.G (400 ng) coding VSV-G protein) by Lipofectamine 2000 transfection reagent. The culture medium was replaced with a maintenance medium at 12 h after transfection. After an additional 36 h, the recombinant virus-containing supernatants were filtered by 0.22-μm PES filter and used to infect A549 cells three times in the presence of polybrene (8 μg/mL) for higher transduction efficiency. 4 days after the first transduction, cells were selected by incubating with 1 μg/mL puromycin for another 10 days, and then the single cell was isolated by serial dilutions and allowed to expand for 2 to 3 weeks without puromycin. Genomic DNA from the cell lines was extracted by using a TIANamp genomic DNA kit (Tiangen, DP304). The modified regions were amplified using specific genomic cleavage detection primers. PCR production was ligated to T-vector by using 5 × TA/Blunt-Zero Cloning Kit (Vazyme, C601-01), followed by transforming into NEB stable bacteria. At least 10 bacteria clones of each transforming were defined by Sanger sequencing and A549 cell lines exhibiting frameshift mutations at the corresponding sites were selected and further confirmed by Western blotting. Two isogenic cell lines bearing the desired gene editing outcomes and one control cell line (NT) were selected for indicated assay. sgRNA sequences and specific genomic cleavage detection primers were listed in Supplementary Table 3.