Extended Data Fig. 2. HUSH-mediated repression of cDNAs and ORF2 fragments correlates with length of the transgene.
a, Western blot showing TASOR depletion in TASOR KD HeLa cells. b, Schematic of assay for the establishment of silencing of lentiviral transgenes. c, Schematic of the gating strategy in ‘one pot’ assay for establishment of silencing. mCherry+ WT and mCherry- TASOR KO HeLa cells were defined based on the mCherry signal and the GFP signal for each of these subpopulations is subsequently plotted on the histogram. d, HUSH-mediated repression of the lentivirus encoding fusion of endonuclease dead Cas9 and KRAB domain (dCas9-KRAB) in HeLa (left) or Jurkat cells (right) measured by flow cytometry. mCherry fluorescence reports mRNA levels from the reporter. For Jurkat cells, a sgRNA targeting the TSS of TASOR was used to deplete TASOR. e, HUSH-mediated repression monitored at different time points post infection and after selection with the antibiotic for the transgene-delivered antibiotic resistance gene. (f) HUSH-mediated repression monitored 48h after transduction of HeLa WT and TASOR KD with lentiviral reporter at different range of MOI. e and f were repeated with different reporters with similar results. g, Scatter plot illustrating a significant correlation between HUSH-mediated repression and length of the insert sequence in the GFP reporters. Each point represents a reporter with different cDNA sequence. Pearson correlation r = 0.7115, two-sided p = 0.0003; 95% CI [0.40 to 0.87] h, Expression of GFP non-coding lentiviral reporters bearing different short cDNA sequences in WT and TASOR KD HeLa cells measured by flow cytometry 72h post transduction. i, HUSH-mediated repression of GFP bearing the indicated untranslated ORF2 fragments measured by flow cytometry. j, Quantification of the HUSH-mediated repression of GFP untranslated reporters bearing full length ORF2 or ORF2 fragments, n = 3 biological replicates ±SD (left). k, RT-PCR analysis of transcripts from GFP reporters bearing ORF2 fragments with primers flanking ORF2 fragments (right). Product sizes corresponding to full length transcripts are 1.7 kb and 3.8 kb for reporters with 1-4 fragments and ∆1-4 fragments respectively.