Extended Data Fig. 5. HUSH knockout is synthetic lethal due to derepression of the LINE-1 transgene.

(a) Gene screen ranks by Z_s scores. HUSH genes are in blue. (b) HUSH complex sgRNA performance during the screen. All knockouts drop out early from LINE-1(+) cells (red) and do not affect growth of luciferase(+) cells (black). Median values are depicted with 95% Confidence Intervals. (c) 12-day clonogenic growth assay in cells expressing LINE-1 (doxycycline-induced) with targeted knockouts of HUSH components compared to non-targeting-control (NTC). n=3 independent experiments. Error bars indicate ±SEM. P values calculated by one-sided T test. (d) Western blot comparing ORF1p and ORF2p expression in HUSH knockout cells or non-target-controls (NTC) that have not been treated with doxycycline compared to NTC with 24 hours of 1 μg/ml doxycycline treatment. ORF1p and ORF2p protein expression are only detected in NTC-treated cells with doxycycline added to the culture media. The double banding pattern for ORF1p is consistently seen with codon-optimized LINE-1. (e) Western blot comparing ORF1p and ORF2p expression 24 hours after 1 μg/ml doxycycline treatment in HUSH knockouts compared to NTC. The ORF2p antibody cannot distinguish between endogenous or transgenic LINE-1 expression. (f) qRT-PCR analysis of LINE-1 transgene expression in HUSH knockouts compared to NTC (induced with 1 μg/ml doxycycline). Because the LINE-1 transgene is codon-optimized, qRT-PCR is specific for the transgene and does not amplify endogenous LINE-1 sequences. *p < 0.001. (g) Linear regression plot of LINE-1 transgene expression and ORF1p and ORF2p protein expression in HUSH knockouts compared to NTC. Shaded area indicates 95% confidence interval for regression line. Both ORF1p and ORF2p increase in expression with higher transgene mRNA expression, although the increase in ORF1p is minimal compared to that observed with ORF2p. (h) Heatmap of immunofluorescence imaging depicting the proportion of cells expressing ORF1p and ORF2p at different levels in HEK293T cells expressing Tet-On LINE-1 (pDA055) at increasing doses of doxycycline.