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sgRNAs targeting the Nanog, Esrrb, Klhl13, and Dusp9 locus were designed using the CRISPR-Cas9 online tool http://crispr.mit.edu:8079/. Off-target scores based on in silico quality and off-target predictions [83] were compared among the candidate sgRNAs and only the top-scoring ones were selected.

CRISPRa sgRNA sequences targeting the TSS of the Dusp9, Cct3, Larp1, Peg10, and Scml2 genes were extracted from previously published libraries [84], whereas sgRNAs targeting the mESC-specific Klhl13 isoform (ENSMUST00000115313.7) were designed using the CRISPR library designer (CLD) from the Boutros lab (Heigwer et al. 2016). sgRNAs targeting the TSS of the Alg13 isoform ENSMUST00000238864.1 were designed using the CRISPOR sgRNA design tool [85].

CRISPRi sgRNA sequences targeting the TSS of the Dusp9, Alg13, Cct3, Larp1, Peg10, and Scml2 were also extracted from previously published libraries [84, 86], whereas sgRNAs targeting the mESC-specific Klhl13 isoform (ENSMUST00000115313.7) were designed using the CRISPOR sgRNA design tool [85]. Safe-targeting control sgRNAs were implemented for multiguide CRISPRa/CRISPRi validation experiments and sequences were extracted from previous publications [87].

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