Blasticidin

J.Lat Full Length Clone 15.4 (Jurkat cells containing near full length HIV-1 expressing GFP) serves as a common HIV-1 latency model for T cells58 (link). We transduced 40,000 of these target cells using 50–250 μl lentiviral supernatants via spinoculation at 1,000 g for 90 minutes at 33 °C. After 2–3 days, we selected for successfully transduced cells using either 2 μg/ml puromycin (Sigma-Aldrich) or 20 μg/ml blasticidin (Bio-connect). Double gRNA expressing cell lines were generated by initial transduction using the appropriate pSicoR-CRISPR-PuroR vector, followed by puromycin selection and subsequent transduction with the appropriate pSicoR-CRISPR-BlastR vector and blasticidin selection. Next, HIV was reactivated from 40,000 transduced cells using 0.1, 1 and 5 ng/ml TNF-α (Life Technologies). After 48 hours, GFP expression was analyzed by flow cytometry.

We transduced 40,000 SupT1 cells using 50–250 μl lentiviral supernatants via spinoculation at 1,000 g for 90 minutes at 33 °C. After 2–3 days, we selected for successfully transduced cells using either 2 μg/ml puromycin (Sigma-Aldrich) or 20 μg/ml blasticidin (Bio-connect). Double gRNA expressing cell lines were generated by initial transduction using the appropriate pSicoR-CRISPR-PuroR vector, followed by puromycin selection and subsequent transduction with the appropriate pSicoR-CRISPR-BlastR vector and blasticidin selection. Next, these transduced SupT1 cells were infected for 2 hours at 37 °C with our reporter virus HIV-1 NanoLuc using an MOI of 0.003 or 0.006. For two months, cultures were monitored daily for CPE and twice a week half of the culture was replaced by fresh culture media. We monitored CPE instead of the potentially more sensitive analysis of luciferase, because we noticed that luciferase could still be expressed from defective proviral DNA, even if we added T20 (1 μM) as a control. As such analysis of luciferase was not a good measurement of ongoing (low level) viral replication and escape. When full-blown CPE was observed, cell free virus was harvested. Viral escape at the gRNA target sites was analyzed as described above (Analysis of viral breakthrough in SupT1 cells expressing a single gRNA).

We transduced 40,000 SupT1 cells using 50–250 μl lentiviral supernatants via spinoculation at 1,000 g for 90 minutes at 33 °C. After 2–3 days, successfully transduced cells were selected with either 2 μg/ml puromycin (Sigma-Aldrich) or 20 μg/ml blasticidin (Bio-connect). Next, transduced SupT1 cells were infected for 2 hours at 37 °C with HIV-1 NanoLuc reporter virus using an MOI of 0.006. After infection, the cells were washed twice and 60 μl of culture supernatant was isolated and stored at −20 °C on a daily basis for subsequent luciferase activity assays. When full-blown CPE was observed, cell free virus was harvested. After DNase treatment of the cell free culture supernatant, viral RNA was isolated and used to investigate viral escape at the gRNA target site. In brief, 200 μl of culture supernatant was treated with 2 μl TURBO DNase (Life Technologies) for 90 minutes in Turbo buffer at 37 °C, after which viral RNA was isolated59 (link). Viral RNA was converted into cDNA and amplified using a one-step RT-PCR reaction (SuperScript^® III One-Step RT-PCR System with Platinum^® Taq DNA Polymerase) (Invitrogen) and on-target primers with specific 454 linker and barcode sequences. The PCR product was subsequently purified using the GeneJET PCR Purification Kit (Thermo Scientific).