Fig. 1: A genome-wide sgRNA screen for SHIN silencing identifies Morc3.

a Setup of the sgRNA screen. T90 ES cells containing an inducible SHIN reporter²⁰ were transduced with a genome-wide sgRNA library pool. Cells were selected for sgRNA vectors and the reporter was activated with doxycycline. Two independent cell batches were cultured to harvest non-selected cells representing the control, and zeocin-resistant cells representing cells with impaired heterochromatin on the SHIN reporter. b Dot plot showing the sgRNA screen results ordered by p-value rank (RIGER SecondBestRank scoring). Major ERV silencing factors are indicated. Morc3 represents a top hit in the screen. c Schematic of the SHIN silencing maintenance assay. T90 ES cells were transduced with sgRNAs for selected candidates and treated for integration with puromycin. Subsequently, doxycycline was added to the cells to induce reporter activity. EGFP expression was analyzed by FACS. d FACS plots depicting EGFP fluorescence in cells with activated SHIN reporter. Almost no activity was detected in cells transduced with a control sgRNA, demonstrating full SHIN silencing. sgRNAs targeting Setdb1 or Morc3 result in SHIN de-repression as indicated by cells showing EGFP expression. e Bar plot depicting the results of the SHIN maintenance silencing assay with selected candidate genes. The red dotted line indicates the background reporter activity. Data are presented as mean values +/− SD of biological replicates per sgRNA (n = 2–4, for details see “Statistics and reproducibility” section). f Schematic of the SHIN silencing initiation assay. T89 ES cells were transduced with sgRNAs for selected candidates and treated for integration with puromycin. Subsequently, the cells were transduced with a control virus without the SHIN sequence or the SHIN reporter with a constitutive promoter, respectively. EGFP expression was analyzed by FACS and the percentage of EGFP expressing cells, relative to the control reporter was calculated. g Bar plot depicting the results of the SHIN initiation silencing assay with selected candidate genes. The red dotted line indicates the background reporter activity. Data are presented as mean +/− SD of biological replicates per sgRNA (n = 1–12, for details see “Statistics and reproducibility” section).