Lenticrispr v2

IAV (A/Puerto Rico/8/34 [H1N1, PR8]) was obtained from the Virus Resource Center of the Wuhan Institute of Virology of the Chinese Academy of Sciences and propagated by allantoic inoculation of 9- to 11-day-old chicken embryos with diluted (1:10⁶) seed virus. Stock virus titers were determined by plaque assays in MDCK cells.
Expression plasmids for DAI-TurboID-Flag, RIPK3, Flag-RIPK3, SPAG94-3xFlag, DAI, and HA–caspase-6 were constructed by inserting the coding sequences into plasmid pCDNA3.1(−), whereas the expression plasmid for HA-RIPK1 was constructed by inserting the coding sequence into pCAGGS. Plasmids pCDNA3.1(−) and pCAGGS were kind gifts from Hanzhong Wang (Wuhan Institute of Virology, CAS). pRK-HA-caspase-8 was kindly provided by Ke Peng (Wuhan Institute of Virology, CAS). The target sequences for single guide RNA (sgRNA)-SPAG9 (humans), sgRNA-SPAG9 (mice), sgRNA-DAI (mice), and sgRNA-RIPK3 (mice) were as follows: 5-TGTGAGAAAGATGTGCTGCA-3′, 5-TTGAACCTTTATATCCACTG-3′, 5-AGTCCTTTACCGCCTGAAGA-3′, and 5-AGTTCTTCACGGCTCACCAG-3′. The sgRNA was cloned into lentiCRISPR-v2 (Addgene 52961). Lentiviruses were produced in HEK293T by transfection of lentiCRISPR v2 together with psPAX2 (Addgene 12260) and pMD.2G (Addgene 12259).

CRISPR‐Cas9‐mediated knockout experiments were performed as previously described (Ran et al, 2013 (link)). In brief, the indicated guide RNA sequences were annealed and subcloned into lentiCRISPRv2 (Addgene, Cat#52961). The cloning was verified by Sanger sequencing. For lentiviral production, 293T cells were seeded into 15 cm plates at 90% confluency 1 day before transfection. The lentiCRISPRv2 vectors (30 μg) containing respective guide RNAs were transfected to the cells together with 20 μg of psPAX2 (Addgene, Cat#8454) and 10 μg of pCMV‐VSV‐G (Addgene, Cat#12260) using PEI. Lentiviruses were harvested and concentrated with PEG Virus Precipitation Kit (Abcam, Cat#ab102538) according to the manufacturer's protocol. The target cells were seeded into 24‐well plates at 20–40% confluency and lentivirally transduced with the concentrated viruses at a 1:25 ratio in antibiotic‐free DMEM supplemented with 10 μg/ml Polybrene (Sigma Aldrich). For the elimination of untransduced cells, puromycin (2 μg/ml) was added to the growth medium 3 days after the transduction. Knockout efficiencies were determined with western blotting. Due to the conclusive roles of TSG101 in cell division and development, a double knockout cell line was not established after single‐cell expansion experiments.

HK-2 cells were transfected with a single guide RNA (sgRNA) specific to ATF4 and XBP1 and designed using the https://cctop.cos.uni-heidelberg.de website. Plasmids expressing Cas9 and sgRNA were generated by inserting custom sgRNA into lentiCRISPRv2 (#52961; Addgene, Watertown, MA, USA) following the manufacturer's instructions. Briefly, lentiviruses were packaged in HEK 293T cells by transfection with specific sgRNA/Cas9-expressing lentiCRISPRv2 and the packaging plasmid psPAX-2 (#12260; Addgene) and psPMD2g (#12259; Addgene) at 1:1:1 molar ratio using the Lipofectamine 3000 Reagent (Invitrogen). Medium was replaced after 6 h and virus samples collected at 24, 48 and 72 h. HK-2 cells were infected with lentivirus in the presence of polybrene (8 μg/ml) and further selected with 1 μg/ml of puromycin to obtain single clones. sgRNAs and primers are listed in Table S2.

pEGFP-C1-RAB40C and the indicated mutants G28N, Q73L and SOCSm (LPLP_212-215to AAAA) were constructed as previously described [12 (link)]. pNTAP-RAB40C, pmDsRed-RAB40C and mutant sequences containing G28N, Q73L and SOCSm were sub-cloned to pNTAP-A vector and pmDsRed-C1, respectively, using the restriction enzymes of EcoR I /Sal I. LentiCRISPRv2 (#52961) was purchased from Addgene^™, and the LentiCRISPRv2-RAB40C_ gRNA1 & 2 were cloned by enzyme BsmBI according to lentiGuide oligo cloning protocol of the Zhang Lab [31 (link)].