Blasticidine s hydrochloride

To generate the NLS‐RNase L, NLS‐RNase L‐R667A, DCP1‐RNase L, and DCP1‐RNase L‐R667A lentiviral particles, HEK293T cells (T25 flasks) were co‐transfected with 2.7 μg pLenti‐EF1‐cmycNLS‐RNase L‐Blast, pLenti‐EF1‐cmycNLS‐RNase L‐R667A‐Blast, pLenti‐EF1‐DCP1‐RNase L‐Blast or pLenti‐EF1‐ DCP1‐RNase L‐R667A‐Blast, 0.8 μg pVSV‐G, 0.8 μg pRSVRev, and 1.4 μg pMDLg‐pRRE using lipofectamine 2000 (Thermo Fisher). Medium was replaced 6 h post‐transfection. Medium was collected at 48 h post‐transfection and filter‐sterilized with a 0.45‐um filter. To create NLS‐RL‐WT and NLS‐RL‐CM A549 stable cell lines, A549 cells were seeded in T‐25 flasks, when 80% confluent, cells were incubated with 1 ml of NLS‐RNase L or NLS‐RNase L‐R667A lentiviral particles containing 10 μg of polybrene for 1 h with periodic rocking. Normal medium was then added to the flask and incubated for 24 h. Medium was removed 24 h post‐transduction and replaced with selective growth medium containing 5 μg/ml of Blasticidine S hydrochloride (Sigma‐Aldrich). Selective medium was changed every few days, then replaced with normal growth medium after 7 days. DCP1‐RNase L and DCP1‐RNase L‐R667A A549 RL‐KO stable cells lines were made in a similar manner.

Blood cells from 20 3rd instar larvae were isolated from each genotype, cultured in a 96-well plate, and allowed to adhere overnight. The next day, the culture medium was substituted with a selective medium containing 10 μg/mL blasticidin (Blasticidine S hydrochloride, Sigma-Aldrich, St. Louis, MO, USA, CatNo. 15205). Cells were cultured for 5 days, with the selective medium replaced every second day. For the Hml>GFP + Hml-DsRed co-culture experiment, blood cells from 10 Hml-DsRed larvae were mixed with blood cells from either 10 Hml>GFP larvae or 10 Hml>GFP>BsdR larvae, and the Hml>GFP>BsdR + Hml-DsRed culture was treated with blasticidin according to the protocol mentioned above.

Lentiviral guide RNA plasmids were constructed with the pLKO.sgRNA.EFS.tRFP (Addgene #57823) backbone. Two small guide RNAs (sgRNAs) were designed for each of RAN, XRCC6, and PSMB4 and were inserted into the vector using BsmB I. The two sgRNAs were used simultaneously except for the functional studies of RAN and PSMB4 knockout. The sequences of the sgRNAs used are in Additional file 8: Table S7.
To produce Cas9-expressing lentiviruses, HEK 293T cells in 100-mm plates with 70% confluency were co-transfected with packaging plasmids pMD2.G (10 μg) and psPAX2 (10 μg), lentiCas9-Blast (Addgene #82372), and pLKO.sgRNA.EFS.tRFP (Addgene #57823), using the calcium-phosphate transfection method. After 12 h, medium was changed to DMEM medium with 10% FBS and 1% PS. Every 12 h thereafter, the culture supernatant containing the viral particles was harvested and subjected to centrifugation at 1700 rpm at 4 °C for 10 min so as to remove any remaining HEK 293T cells.
To generate primary breast cancer cells that stably express Cas9, the culture supernatant containing the viruses was supplemented with polybrene (1 μg/ml) and administered to cancer cells for 12 h. This process was repeated three times. After 24-h incubation in fresh RPMI media, the stable Cas9-expressing cells were obtained by selection with 4~8 μg/ml of Blasticidine S hydrochloride (Sigma).

Retro-, and lentiviral vectors were produced by transfecting HEK293T cells (CRL-3216, ATCC) with one of the following vectors: pMMLV-CAG-SAD B19_optimized_G-IRES-Puro, pMMLV-CAG-optimized_T7pol-IRES-BSD, pLenti-EF1a-envA-IRES-Neo and pMMLV-CAG-TVA-IRES-Puro, along with the compatible retro- or lenti-viral GAG-Pol and the vesicular stomatitis virus glycoprotein (VSV-G), by means of calcium-phosphate precipitation. Viruses were collected and filtered 48 hr post transfection and used to transduce low-passage HEK293T or BHK-21 (CCL-10, ATCC) cells. Three days post transduction, the cells were passaged and one of the following antibiotics were added to the medium in order to select for the cells which stably express the respective construct: Puromycin dihydrochloride (3 µg ml^–1), blasticidine S hydrochloride (15 µg ml^–1), or G418 disulfate (Neomycin, 500 µg ml^–1, Sigma-Aldrich in all cases). Once the cells reached full confluence again, they were passaged, and again supplemented with the antibiotics. This cycle was repeated for at least three times a week for two weeks before initial use for production of rabies viral vectors. The new cell lines have all tested negative for mycoplasma. All cell lines and plasmids presented in this study are available from the corresponding author upon request and all plasmids are also available from Addgene (See Key resources table).

HEK293T (ATCC CRL-11268, research resource identifier [RRID] CVCL_1926), HEK293T-ACE2 (BEI NR-52511, RRID CVCL_A7UK), and Vero E6 expressing high endogenous ACE2 (Vero-ACE2) (BEI NR-53726, RRID CVCL_A7UJ) supplemented with 1% penicillin/streptomycin (MilliporeSigma, P4333) and 10% (vol/vol) fetal bovine serum (FBS) (Thermo Fisher Scientific, 26140-079) were used. Calu-3 cells, a gift of Estelle Cormet-Boyaka at The Ohio State University, were grown in Eagle’s minimum essential medium (EMEM) (ATCC, 30-2003) supplemented with 1% penicillin/streptomycin and 10% (vol/vol) FBS. HEK293T-ACE2 cells or Vero-ACE2 cells stably expressing TMPRSS2 were generated by transduction of pLX304 vectors expressing TMPRSS2 (a gift from Siyuan Ding at the Washington University in St. Louis, MO) and selection by blasticidine S hydrochloride (MilliporeSigma, 15205) (10 μg/mL for HEK293T-ACE2 cells and 7.5 μg/mL for Vero-ACE2 cells) for 7 to 14 days. All cell lines in this study were maintained at 37°C in the presence of 5% CO₂.