HEK.dDHXs cell line generation via CRISPR/Cas9 genome editing

Gene-specific gRNA-encoding oligonucleotides were cloned into the pSpCas9-gRNA-GFP plasmid (Addgene PX458; no. 48138) targeting the C terminus coding region of the endogenous DHX15 and DHX38 genes, using BbsI restriction digestion and ligation. The oligo sequences used for cloning are provided (Table S1). DNA repair template plasmids containing DHX15-FKBP^F36V-2xHA_P2A_BFP, DHX15-FKBP^F36V-2xHA_P2A_HygR, and DHX38-FKBP^F36V-2xHA_P2A_BFP were synthesized with 800bp dsDNA homology arms by Genewiz and assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB E5520S).

To generate the endogenously tagged lines, one million HEK293T.A2 cells were co-transfected with the pSpCas9-gRNA-GFP plasmids and the pUC19-FKBP^F36V-P2A-selection repair template plasmids at 1:1 ratio (Figure S1A). Transfection of HEK293T.A2 cells was performed using Lipofectamine3000 (Invitrogen L3000008) according to the manufacturer’s instructions.

Two days post-transfection, cells were sorted for the expression of Cas9-GFP. Five days post GFP sorting, cells were sorted again for BFP, and serially diluted in 100 µg/ml Hygromycin-containing (Millipore 400052) growth media to allow single-cell clone formation. HEK.dDHX38 line was generated without a second HygR repair template, and the BFP-positive cells were serially diluted to grow in regular growth media. One week post sorting, viable single clone colonies were picked using a stereoscope into a 96-well plate. Two days after clone-picking, 80% of the cells were collected into a 96-well PCR plate for genotyping. For genotyping, cells were lysed in direct PCR reagent (Viagen 301-C) supplemented with 1 μL proteinase K (Viagen 501-PK), and further used as templates in genotyping PCR reactions (NEB OneTaq Quick-Load 2xMasterMix, M0486L) (Figures S1B and S1C). Clones with homozygous degron tags were expanded and used for RNA-seq experiments.