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To generate virus carrying sgRNAs of the GeCKOx and GeCKOxs libraries, HEK293T cells were seeded into 12/8 (for GeCKOx/GeCKOxs) 10-cm plates and transfected the next day at 90% confluence. Each plate was transfected with 6.3 μg of pPL1, 3.1 μg of pLP2, and 2.1 μg of VSVG vectors (Thermo Fisher Scientific) together with 10.5 μg of the GeCKOx/GeCKOxs library plasmids in 1 ml of Opti-MEM (Life Technologies). Sixty microliters Lipofectamine 2000 Reagent (Thermo Fisher Scientific) were diluted in 1 ml Opti-MEM. Both mixtures were incubated separately for 5 min and then combined followed by a 20 min incubation, after which they were added dropwise to the HEK293T cells. Medium was changed 6 h after transfection. Transfected HEK293T cells were cultured for 48 h at 37 °C; afterwards, the medium was collected and centrifuged at 1800×g for 15 min at 4 °C. Viral supernatant was further concentrated 10-fold using the lenti-X™ Concentrator (Takara Bio) following the manufacturer’s instructions and subsequently stored at − 80 °C.

To assess the viral titer, 5 serial 10-fold dilutions of the viral stock were applied to each well of a 6-well mESC plate (MOCK plus 10− 2 to 10− 6) for transduction with 8 ng/μl polybrene (Merck). Two replicates were generated for each well. Selection with puromycin (1 ng/μl, Sigma) was started 2 days after transduction and colonies were counted after 8 days. The number of colonies multiplied with the dilution factor yields the transducing units per ml (TU/ml), which ranged from 0.5–1.5 × 106 TU/ml.

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