Topo ta cloning kit for sequencing

5′ and 3′ PUM1 RACE was performed from H99, and 5′ PUM1 RACE was performed from P_GAL7ZNF2 using GeneRacer^® Kit with SuperScript^® III RT and TOPO TA Cloning^® Kit for Sequencing (ThermoFisher Scientific). RNA from midlog H99, P_GAL7ZNF2 induced for 3 hours in YP-Gal and mating culture from V8 plate was isolated and treated with DNase, and then cDNA was generated using an iScript cDNA synthesis kit9 (link). Reaction mixtures lacking reverse transcriptase were included as a control for DNA contamination. The cDNA was used in a semi quantitative PCR. The two dominant bands obtained were further cloned in pJet1.2 blunt as per the manual (CloneJET PCR Cloning Kit) and subsequently sequenced using pJet1.2 forward and reverse primers.

Genomic PCRs for analysis of mKO2_G67D and SOD1_R115G point mutations were performed using Phusion™ Hot Start II High-Fidelity DNA-Polymerase (Thermo Fisher Scientific #F549L) in HF buffer with 2 ng/μL of respective genomic DNA template, 0.2 mM of each, dATP, dGTP, dCTP and dTTP and 0.2 μM of each primer (mKO2 PCRs: mKO2 gen for + mKO2 gen rev; SOD1 PCRs: SOD1 for #2 + SOD1 rev #2; Primer sequences are listed in Supplementary Table 1). PCR program was set according to manufacturer’s instructions using 64 °C annealing for mKO2 PCRs and 60 °C for SOD1 PCRs with 20 s extension time for mKO2 PCRs and 15 s for SOD1 PCRs, for a total of 35 cycles. For detailed analysis of indels from Cas9 nuclease assays and PE assays (Supplementary Fig. 3), mKO2 PCR products were sub-cloned using TOPO™ TA Cloning™ Kit for Sequencing (Thermo Fisher Scientific #450030) according to manufacturer’s instructions and analyzed by Sanger sequencing.

Constructs for stable constitutive or Dox‐inducible USP22 and RIPK3 expression were generated by cloning Sfi1‐digested full‐length human USP22 and RIPK3 PCR products into pSB‐tet‐Neomycin (Addgene plasmid #60509) pSB‐tet‐Puromycin (Addgene plasmid #60507), pSBbi‐Blasticidin (Addgene plasmid #60526) (Kowarz et al, 2015), using standard procedures and into pT‐Rex‐DEST30 (#12301016, Thermo Fisher Scientific), or into the pcDNA 3.1 (+) (#V79020, Thermo Fisher Scientific) using the TOPO TA Cloning Kit for Sequencing (Thermo Fisher Scientific). All RIPK3 mutants and silent PAM mutations for re‐expression of RIPK3 or USP22 were generated with the Gene Art Site Directed Mutagenesis System Kit (Thermo Fisher Scientific), according to the manufacturer's instructions and subjected to Sanger DNA sequencing for verification. For transient ectopic expression, constructs were transfected in cells using FuGENE HD Transfection Reagent (Promega) or Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. A corresponding amount of EV plasmid was used to adjust total DNA amount, if required. For in vivo ubiquitination assays, a HA‐tagged (Addgene plasmid #17608) or His‐tagged (kindly provided by Stefan Müller, Frankfurt am Main, Germany) ubiquitin construct was used. Primer sequences and further details on cloning are available upon request.

The genomic DNA of single colonies was extracted using QuickExtract DNA extraction solution (Lucigen) following the manufacturer’s instructions. Targeted locus was amplified by PCR using MeltDoctor™ HRM Master Mix (Applied Biosystems) and primers (5′-TGC​TCT​GAG​TTG​TAT​TTG​TGT​TAA​C-3′, 5′-GAG​AGG​TTG​TAA​CTT​ACC​TTT​TCC​A-3′) according to the manufacturer’s instructions. PCR reactions were analyzed using a QuantStudio 12K Flex Real-Time PCR system (Applied Biosystems). The protocol was 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min, followed by 95°C for 10 s and 60°C for 1 min; then, the temperature was increased by 0.025°C/s until 95°C 15 s, followed by cooling to 60°C. Curves were analyzed using QuantStudio™ 12K Flex software. Clones showing editing were selected, and corresponding PCR products were sequenced by Sanger sequencing (Eurofins).
To ensure the purity of each edited clone, corresponding PCR products were subcloned using a TOPO™ TA Cloning™ Kit for Sequencing, with One Shot™ TOP10 Chemically Competent E. coli (Thermo Fisher Scientific) according to manufacturer’s instructions. At least 30 TA clones for each CRISPR cell line were analyzed by Sanger sequencing (Eurofins).

hPSCs were cultured in a well of a 6-well plate until reaching 80% confluency. Once reaching this confluency, genomic DNA was then isolated using the ZYMO Quick DNA Miniprep Plus kit (Zymo Research). This genomic DNA was then used as a template for PCR amplification of genomic regions of interest. PCR was carried out using GoTaq DNA polymerase (Promega) with appropriate primers. The resulting amplicons were run through 1% agarose gels, and bands of interest were gel purified using the Zymoclean Gel DNA Recovery kit (Zymo Research) and subsequently run through the Zymo clean and concentrator-5 kit (Zymo Research). The resulting amplicons were then cloned into the TOPO TA cloning plasmid using the TOPO TA Cloning Kit for Sequencing (Thermofisher) according to the manufacturer’s instructions. The resulting cloned plasmids were finally transformed into One Shot Stbl3 E. coli cells (Thermofisher) according to manufacturer’s instructions, plated on Ampicillin agar plates, and cultured at 37°C overnight. Single E. coli colonies were then picked and cultured in LB broth overnight, cultured at 37°C and shaking at 250 rpm. The next day, plasmids were purified using the Zyppy Plasmid Miniprep Kit (Zymo Research) and sent in for sequencing.

U2-OS cells were treated with AAV-λ465 and were transfected with SpCas9-VRQR variant and gRNA against NGA FANCF site 3 (see details of transfection above). Genomic DNA was isolated by the Qiagen Blood and Tissue Kit, and 100 ng DNA was subjected to targeted PCR (see primers in Supplementary Table 4) using the Phusion polymerase (NEB). The samples were subjected to gel purification (gel was cut above the expected 132 bp PCR band and below ~600 bp). After gel purification, DNA sample was ethanol precipitated for 20 min at −20 °C and 1 μL reaction (12 ng) was used to in the TA cloning reaction according to manufacturer’s instructions (TOPO® TA Cloning Kit for Sequencing, ThermoFisher). Three full plates (285 clones) were sequenced with the T7 primer included in the cloning kit.