Pmdlg

Lentiviral vectors targeting human MDA5 (Ifih1 gene) single guide RNAs (sgRNAs) were obtained from Applied Biological Materials. HEK293T cells were transfected with pRSV-Rev, pMDLg, pVSV-G plasmids (Addgene), and the pLenti-IFIH1 sgRNA using Lipofectamine 2000. Supernatants containing lentiviral particles were collected after 48 h and concentrated through Amicon Ultra-15 filter (100 K, Millipore-Sigma). HeLa cells were then transduced with the Lentiviral vectors by directly adding concentrated supernatant together with polybrene (5.0 μg/mL, Millipore-Sigma) to cells. Polyclonal cells with positive transduction were selected through puromycin (2.0 μg/mL).

Lentiviral plasmids expressing the control (empty), miR-22 mimic, or miR-22 inhibitor sequences were obtained from Biosettia (San Diego, CA, USA). Lentiviruses were prepared in 293 T cells by cotransfection of miRNA-encoding plasmids and helper plasmids such as pRSV-REV, pMDLg, and pVSV-G (Addgene, Watertown, MA, USA). To generate control, miR-22-overexpressing, or miR-22 inhibitor-expressing macrophage cells, RAW 264.7 were infected with lentiviruses and further incubated with puromycin.

Transduction was performed for generating cells expressing the mClover3-FKBP target or for CRISPR KOs (Table S3). mClover3-FKBP was expressed from an EF1a-driven transfer vector (Addgene TBA), and sgRNAs and Cas9 were expressed from lentiCRISPR v2 (Addgene 52961). Virus was generated in HEK293T cells by transfection with the aforementioned transfer vectors and pMDLg (Addgene 12251), pRSV-REV (Addgene 12253), and pMD2g (Addgene 12259). mClover3-FKBP-containing virus was transduced at low efficiency (~10%) as measured by flow cytometry, while lentiCRISPR v2 transduction efficiency was ~50% as measured by cell counting after puromycin selection. Infection was performed in the presence of polybrene (Millipore, TR-1003-G), a day after which the cells were replenished with fresh media. Enrichment was performed 48 hours after infection by fluorescence-activated cell sorting (FACS) or puromycin selection, as appropriate.

Cas9/gRNA RNP VLP was generated as described previously [22 (link)]. Briefly, 293T cells were seeded in a 10 cm dish with DMEM medium supplemented with 10% fetal bovine serum. After 24 h, the culture medium was replaced with 8 ml of Opti-MEM. A total of 7.5 µg of plasmids expressing brachyury targeting gRNAs a + c, b + c, a + d, or b + d (See Fig. 1A) were mixed with 7.5 µg of COM-modified packaging plasmid pspAX2-D64V-NC-COM and 7.5 µg of plasmid pMDLg (Addgene, Watertown, MA, USA) in 600 µl Opti-MEM. 40 µl of Fugene HD was then be added and incubated at room temperature for 10 min. The mixture was added into 293T cells. After 24-h transfection, the culture medium was changed with 15 ml of Opti-MEM. The supernatant containing Cas9 RNP VLP was collected after an additional 48-h transfection and spun for 5 min at 500 g to remove cell debris. The Cas9/gRNA RNP VLP in the supernatant was concentrated by ultracentrifugation at 100,000 g for 2 h at 4 °C. VLP pellets were resuspended in PBS. Cas9/gRNA RNP VLP was quantified by p24 based ELISA according to the manufacturer’s instructions.