Human TRIT1 mutant cells and control cells were generated using the CRISPR/Cas9 system essentially as described previously (Shalem et al. 2014 (link)). Briefly, sense and antisense oligonucleotides encoding a single guide RNA (sgRNA) against TRIT1 gene or a control sgRNA (not targeting human genome) (Supplemental Table 1 ; Shalem et al. 2014 (link)) were cloned into the BsmBI sites of lentiCRISPR v2 Blast plasmid (Addgene #83480). Lentiviruses were generated in HEK293FT cells by transfecting the sgRNA sequence-containing lentiCRISPR v2 Blast plasmid, psPAX2 plasmid (Addgene #12260) and pMD2.G plasmid (Addgene #12259) with Lipofectamine 3000 (Invitrogen). Fresh HEK293FT cells were transduced with the generated viruses, followed by blasticidin selection of the transduced cells. Subsequently, single clones were acquired by diluting the cells in 96-well plates followed by expansion of the clones. The target region of the genome in each clone was PCR-amplified and sequenced with conventional Sanger sequencing, using the primers listed in Supplemental Table 1 .
Pspax2 plasmid
Manufactured by Addgene
Sourced in United States
PsPAX2 is a plasmid that contains the necessary packaging genes for the production of lentiviral particles. It provides the structural and enzymatic components required for viral genome packaging without the inclusion of the viral genome itself.
Automatically generated - may contain errors
Lab products found in correlation
Pmd2 g plasmid, by Addgene (14 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Polybrene, by Merck Group (5 mentions)
Pmd2 g, by Addgene (3 mentions)
Pcmv vsv g plasmid, by Addgene (2 mentions)
Vsv g plasmid, by Addgene (2 mentions)
Puromycin, by Merck Group (2 mentions)
Polyethylenimine (pei), by Merck Group (2 mentions)
Lenticrisprv2 blast plasmid, by Addgene (1 mentions)
Hepes, by Merck Group (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Lenticrispr v2, by Addgene (1 mentions)
Plenti cmv puro dest w118 1 vector, by Addgene (1 mentions)
Plvx ires zsgreen1 vector, by Takara Bio (1 mentions)
Pei 25k, by Polysciences (1 mentions)
Plko 1 vector, by Addgene (1 mentions)
Lipofectamine 2000, by Addgene (1 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
25 protocols using pspax2 plasmid
1
CRISPR-Mediated TRIT1 Knockout Generation
2
HCMV US33as-5p Knockout Using CRISPR-Cas9
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hcmv-33as-5p was knocked out with the lentiCRISPR v2 plasmid as described previously (16 (link), 17 (link)). Briefly, sgRNAs targeting hcmv-US33as-5p sequences capped by a 5′-N20GG PAM sequence was designed using an online tool (https://zlab.bio/guide-design-resources ). The sgRNAs were then synthesized and annealed and ligated to the linearized vector. Lentivirus was produced at a high titer in 293 cells by co-transfection with 2.6 μg of CRISPRv2 vector, 0.25 μg of pVSVg plasmid (Addgene 8454), and 2.5 μg psPAX2 plasmid (Addgene 12260). MRC-5 cells were infected with the Cas9/sgRNA lentivirus (MOI = 0.5), and stable transfectants were selected using puromycin (2.5 μg/mL). The MRC-5 cells were then infected with HCMV (Toledo strain, MOI = 1), and the supernatant, which contained the mutant virus, was collected at 72 hpi. The mutant virus was seeded in 96-well plates containing MRC-5 cells for plaque assays and isolated as described previously (18 (link)).
3
Lentiviral Transduction of Rac1 in HEK293T
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HEK293T cells, grown 4 days in media without antibiotics and at 50–70% confluency, were co-transfected using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) with the pMD2.G plasmid, the psPAX2 plasmid (both from Addgene, Watertown, MA, USA) and the pLOC RAC1 wild-type plasmid at a 1:3:4 ratio, respectively. Six hours later, media was changed to media with 1M HEPES, 0.85%NaCl (Lonza, Houston, TX, USA) added to a final concentration of 25 mM, pH 7.2–7.5. The supernatant was collected in 1 mL aliquots 24, 48, and 72 h later and frozen at −80 °C. For transductions, polybrene (EMD Millipore, Burlington, MA, USA) was added to media containing 25 mM HEPES final as above, and the media mixture added 1:1 to thawed virus. Then, the final mixture was added to ~70% confluent KTC1 cells following manufacturer’s instructions. One day later, the media mixture with the virus was removed and replaced with fresh culture media. Cells were expanded in D-MEM/F12 culture media containing 10% FBS and 30 µg/mL blasticidin.
4
Lentiviral Transduction of PANC-1 Cells
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Lentivirus was generated in HEK293T cells (ATCC, Manassas, VA) in 225-cm2 flasks. Briefly, 22.2 μg of a CRISPR pooled gRNA library (human sgRNA library Brunello in lentiGuide-Puro) transfer vector (Addgene, cat. #73178) [17 (link)], 16.7 μg of psPAX2 plasmid (Addgene, cat. # 12260), and 11 μg of pMD2.G plasmid (Addgene, cat. # 12259) were combined with Lipofectamine 3000 (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol, and the mixture was used to transfect the cells. The virus-containing medium was collected 48 h after transfection and centrifuged at 500×g for 5 min to remove cells and debris. The supernatant containing the virus was then filtered with a 0.45-μM PES filter and frozen at − 80 °C. Viral transduction was accomplished by adding 150 μL of virus-containing medium per 1 × 106 cells (titer determined experimentally, MOI = 0.3) to 225-cm2 flasks of PANC-1 cells at 75% cellular confluence, along with 4 μg/mL polybrene (Sigma-Aldrich), for 16 h. The virus-containing medium was then replaced with fresh growth medium.
5
Generation of CRISPR-Cas9 Knockout Cell Lines
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We generated hHB−/−, RIG-I−/−, and MDA5−/− cells using a lentivirus-expressing CRISPR-Cas9 vector (lentiCRISPRv2; Addgene). The hHB-, RIG-I-, and MDA5-specific subgenomic RNA (sgRNA) sequences were the following: for hHB, 5′- GTA ACG GCA GAC TTC TCC TC-3′ (forward) and 5′-GAG GAG AAG TCT GCC GTT ACC-3′ ( reverse); for RIG-I, 5′-GGG TCT TCC GGA TAT AAT CC-3′ (forward) and 5′-GGA TTA TAT CCG GAA GAC CCC-3′ ( reverse); and for MDA5, 5′-CGA ATT CCC GAG TCC AAC CA-3′ (forward) and 5′-TGG TTG GAC TCG GGA ATT CGC-3′ ( reverse). Lenti-CRISPR virions were packaged in HEK293T cells by transfecting the psPAX2 plasmid (Addgene), the pMD2.G plasmid (Addgene), and either the lentiCRISPRv2 vector containing hHB-, RIG-I-, or MDA5-specific sgRNA or an empty lentiCRISPRv2 plasmid as a control. The suspensions were harvested at 72 h posttransfection (hpt). HEK293T cells were infected with the suspensions and treated with 1.5 μg/ml puromycin for 5 days. The cells were lysed, and hHB, RIG-I, or MDA5 expression was analyzed by Western blotting.
6
Lentiviral Transduction of T Cells with MDA-7/IL-24
7
Lentiviral Knockdown and Overexpression
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The following short hairpin RNA (shRNA) expression sequences were used: DDX52, 5′-GCCAATCCAAATGCAAGCCAT-3′; c-Myc, 5′-CCCAAGGTAGTTATCCTTAAA-3′; and control, 5′-GCTCCGTGAACGGCCACGAGT-3′. These sequences were cloned into the PLKO.1 vector (10879; Addgene, Watertown, MA, USA). The overexpressed DDX52 plasmids were cloned into the pLVX-IRES-ZsGreen1 vector (Takara Bio, Shiga, Japan) via DNA assembly (#E5520; NEB, Rowley, MA, USA).
The psPAX2 plasmid (12260; Addgene) and pCMV-VSVG plasmid (8454; Addgene) in 293 T cells were transfected into HEK293T cells with PEI 25K (23966-1; Polysciences, Warring, PA, USA) following the manufacturer’s instructions. The culture medium was collected after 48 and 72 h, and the supernatant was centrifuged at 1000 rpm for 10 min to obtain the supernatant products containing the lentivirus to transduce into the indicated cells. Puromycin (5 µg/ml) (Sigma–Aldrich, St. Louis, MO, USA) was used to isolate the stable transformants in PC3 and 22RV1 cells.
The psPAX2 plasmid (12260; Addgene) and pCMV-VSVG plasmid (8454; Addgene) in 293 T cells were transfected into HEK293T cells with PEI 25K (23966-1; Polysciences, Warring, PA, USA) following the manufacturer’s instructions. The culture medium was collected after 48 and 72 h, and the supernatant was centrifuged at 1000 rpm for 10 min to obtain the supernatant products containing the lentivirus to transduce into the indicated cells. Puromycin (5 µg/ml) (Sigma–Aldrich, St. Louis, MO, USA) was used to isolate the stable transformants in PC3 and 22RV1 cells.
8
Lentiviral Vector Production in HEK293T Cells
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HEK293T cells were cultured in individual 15 cm dishes for each viral construction. When the cells reached approximately 70–80% confluency (~ 1.5 × 107 cells), they were cotransfected with 15 μg of the pLVX-LAT2 plasmid, 15 μg of the psPAX2 plasmid (Addgene #12260, Cambridge, MA, USA) and 10 μg of the pMD2.G plasmid (Addgene #12259, Cambridge, MA, USA) plasmid with Lipofectamine 2000 transfection reagent. At 18 h post-transfection, the medium was replaced with 25 mL of fresh complete medium. The supernatants were harvested at 48 h and 72 h, centrifuged at 1000 rpm for 10 min at 4 °C to remove the cells, and filtered through a 0.45 μm filter to remove the debris. Finally, the supernatant was ultracentrifuged at 120,000×g for 2 h at 4 °C, dissolved in PBS after the removal of the supernatant, and stored at − 80 °C.
9
Engineered U2AF1 Protein Expression
10
Lentiviral Vector Construction for Bcl-XL Expression
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The lentiviral vector lentiCRISPR [55 (link)] was obtained from Addgene (#868, Addgene, ref. no. 52961). The pMD2.G plasmid (#554, Addgene, ref. no. 12259) encodes the envelope of lentivirus. The psPAX2 plasmid (#842, Addgene, ref. no. 12260) encodes the packaging system. LeGOiG2-Bcl-XL (#863) was constructed by subcloning the 771 bp EcoRI fragment from hBcl-XL.dn3 (#274) into LeGOiG2 (#807; Addgene: plasmid 27341).
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