Generation of Dgcr8_KO and Drosha_KO mESCs using CRISPR/Cas9

Dgcr8_KO and Drosha_KO mESCs were generated from E14TG2a mESCs using a paired CRISPR/Cas9 strategy (Wettstein et al., 2016). The plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (plasmid 42230; Addgene) was used. Cells were single-cell sorted 48 h after transfection using a flow cytometry cell sorter (MoFlo; BD) into 96-well plates (one single cell per well). The first screening for selection of candidates was performed at the genomic level by PCR. All of the primers used for CRISPR/Cas9 constructions and PCR screening are described in Table S1. Specific CRISPR/Cas9 sgRNAs were generated using E-CRISPR software (Heigwer et al., 2014) or alternatively chosen from an established library (Koike-Yusa et al., 2014). All designs are based on the latest mouse genome assembly (GRCm38/mm10) provided by the University of California, San Cruz, Genome browser (http://genome.ucsc.edu/).