Pdonor d09

Oligonucleotides targeting TDP-43 were ordered from Integrated DNA Technologies (Integrated DNA Technologies, Coralville, IA, USA). Oligonucleotides were annealed and cloned into pCas-Guide (Origene, Rockville, MD, USA) according to manufacturer’s protocol. gRNA targeted the following sequence: GTTTGTGGGGCGCTGTACAG. Cloned pCas-Guide was co-transfected with pDonor-D09 (GeneCopoeia, Rockville, MD, USA) using Lipofectamine 2000 (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) into U-2 OS cells. The following day, cells were treated with puromycin (1.5 μg/mL). After 48 h of selection, puromycin was removed. Single-cell clones were generated by limiting dilution.

The gRNA sequence (5′-ATTGGTTTAATAACGTCCCC-3′) was cloned between the BamHI site and the BsmBI site of the pCas-Guide vector (OriGene, Rockville, MD, USA). Sequences of the homology arms flanking the PAM site were cloned into pDonor-D09 (GeneCopoeia, Rockville, MD, USA). U2OS cells co-transfected with these plasmids were cultured for 14 days with puromycin and screened for loss of USP42 expression by immunoblotting. To obtain complemented cell lines, the USP42 KO cell line was transfected with the plasmids encoding GFP or GFP-USP42 (FL, ΔC or Δ946–1196) and cultured for 14 days with 1 mg/ml Geneticin.

ANG knockout and RNH1 knockout U2OS cells were generated using CRISPR/Cas9-mediated gene editing as previously reported (Lyons et al., 2016 (link)). Briefly, oligonucleotides corresponding to a gRNA targeting the sequence 5′- TGG​TTT​GGC​ATC​ATA​GTG​CT-3′ in ANG and 5′-GAG​CCT​GGA​CAT​CCA​GTG​TG-3′ in RNH1 were designed using CRISPR Design software from the Zhang lab (crispr.mit.edu). Oligonucleotides were annealed and cloned into the pCas-Guide vector (Origene) according to the manufacturer’s protocol, and the resulting plasmid was co-transfected with pDonor-D09 (GeneCopoeia), which carries a puromycin resistance cassette, and into U2OS cells using Lipofectamine 2000 (Invitrogen). The following day, cells were selected with 1.5 μg/ml of puromycin for 24 h only, to lessen the likelihood of genomic incorporation of pDonor-D09. Cells were cloned by limiting dilution, and screened by western blot analysis using anti-ANG antibody (Santa Cruz, C-1) and anti-RNH1 antibody (Proteintech group, 10345-1-AP). Anti-beta-actin antibody (Proteintech group, 66009-1-Ig) was used as a loading control. Knockout was confirmed by genotyping and western blot analysis (Supplementary Figure S1).