Publication protocol
"293T cells were plated at 2 × 105 cells per well in 6-well plates and transfected using the calcium phosphate method (Cullen 1987) with 2 µg of the Cas9/sgRNA expression plasmid, 200 ng and 800 ng of the CD4 and CXCR4 receptor expression plasmids (Tokunaga 2001). In parallel, 2 × 106 293T cells per 10 cm dish were transfected with 10 µg of the proviral indicator clone pNL-NLuc-HXB using PEI. 72 hpt the supernatant medium from the HIV-1 producer cells was filtered through a 0.45 µM filter, treated with DNase I (NEB) for 1 hour at 370C, and then used to infect 2 × 105 Cas9 expressing 293T cells. Similarly, in the case of Cas9-expressing SupT1 cells, 2 × 105 transduced cells were infected with the NL-NLuc-HXB virus in 48-well plates. The infected cells were then incubated for an additional 24 h for 293T and 72 h for SupT1 cells, prior to harvest and analysis of the NLuc expression level.
For analysis of viral replication in the presence of Raltegravir (Sigma Aldrich), a 10 mM stock was initially prepared in DMSO. 293T cells were then pretreated with either Raltegravir (500:1 dilution to give a final concentration of 20µM), or an equal volume of DMSO alone, 24 hours prior to infection. Infections were then performed in the presence or absence of Raltegravir for the duration of the experiment.
For analysis of TAR DNA editing efficiency, DNA was harvested from Cas9- and sgRNA-expressing cells infected with NL-NLuc-HXB using the Quick-DNA Plus Kit (ZYMO). PCR products were generated by nested PCR using primers LTR-F1 and HIV-R1, followed by a second PCR reaction using primers LTR-F1 and HIV-R2 (see Supp. Table 2). PCR products were purified using GeneJet columns (Thermo-Fisher),digested with restriction enzymes BglII or SacI (NEB) and analyzed by agarose gel electrophoresis. Images were captured using GeneSnap and quantified using GeneTools (Syngene). To identify any mutations introduced into TAR by Cas9, PCR primers LTR-F1 and HIV-R1 (Sup. Table 2) were used to isolate segments of the HIV-1 TAR region that were then cleaved with EcoR I and Xho I, cloned into pcDNA3 and then sequenced using the T7 primer.
For RNA analysis, cells were lysed in TRIzol (Thermo Fisher Scientific) and RNA harvested according to the manufacturer’s instructions. The RNA was then converted to DNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). PCR products for analytical digestion were generated by nested PCR using primers TAR-F and HIV-R3, followed by gel purification and a second PCR using primers TAR-F and HIV-R4 (Sup. Table 2). The final DNA product was purified using GeneJet columns (Thermo-Fisher), digested with SacI and then analyzed on a 2% agarose gel. Images were captured using GeneSnap and quantified using GeneTools."
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