CRISPR-Cas9 Genome Editing in SH-SY5Y Cells

The single‐guide RNAs (sgRNAs) used in this study were purchased from Integrated DNA Technologies (IDT). These modified sgRNAs contain 2′‐O‐methyl‐3′‐phosphorothioate modifications at the three terminal nucleotides of the 5′ and 3′ ends. Ribonucleoprotein (RNP) complexes were made by mixing 100 pmol sgRNA and 6 μg Cas9 nuclease V3 protein (IDT) in 5 μl Opti‐MEM (Gibco) for incubation at room temperature for 25 min before electroporation. SH‐SY5Y cells (300,000 cells) resuspended in 15 μl Opti‐MEM were mixed with the RNP complexes and subjected to electroporation using the Lonza 4D electroporator (program CM138). The gene knockout efficiency was validated by Western blotting. The sgRNA spacer sequences were as follows:
AAVS1 sgRNA: 5′‐GGGGCCACUAGGGACAGGAU‐3′
GSDME sgRNA 1#: 5′‐AAGUCCGACUCCACGACCAC‐3′
GSDME sgRNA 2#: 5′‐CUCCUCCAUUCCAGUGGUCG‐3′
Caspase 2 sgRNA 1#: 5′‐UGUAGGAUAUUGGGAGUGUG‐3′
Caspase 2 sgRNA 2#: 5′‐UUUAGAGUUUCCUGAUGAUG‐3′
BiD sgRNA 1#: 5′‐UCAACAACGGUUCCAGCCUC‐3′
BiD sgRNA 2#: 5′‐GAUGCACUCAUCCCUGAGGC‐3′
Caspase 3 sgRNA 1#: 5′‐CUAAACAGAAAGAUCAUACA‐3′
Caspase 3 sgRNA 2#: 5′‐GGAAGCGAAUCAAUGGACUC‐3′
Caspase 7 sgRNA 1#: 5′‐GCCCUGAUCAUCUGCCAUCU‐3′
Caspase 7 sgRNA 2#: 5′‐UCCCAGAUGGCAGAUGAUCA‐3′
IFI16 sgRNA 1#: 5′‐ACUGACCACAAUCAACUGUG‐3′
TRIF sgRNA: 5′‐CGAAGGCGCUAGGAAGUGAU‐3′
MAVS sgRNA: 5′‐AGGUGGCCCGCAGUCGAUCC‐3′
MYD88 sgRNA: 5′‐CUGUCUCUUCCCCACAGAGG‐3′
STING sgRNA: 5′‐CAGUCCUCCAGUAGCUGCCC‐3′
IRE1α sgRNA 1#: 5′‐UUCAGGAAGCGUCACUGUGC‐3′
IRE1α sgRNA 2#: 5′‐CAGCGUUGACACAAACAACA‐3′.