pAAVS1-TLR6 was constructed by generation of a 1552 bp fusion PCR product from a 329 bp PCR fragment (using primers TLRvenus-1 and TLR6–1) and a 1247 bp PCR fragment (using primers TLR6–2 and TagRFP-3), using pAAVS1-TLR donor (Addgene ID 64215)  as template, into the backbone of plasmid pCAG-venusTarget+1P2A + 3TagRFP (opened with PacI and MluI), resulting into pCAG-TLR6. pCAG-TLR6 was used for isolation of a 3.2 kb AscI-AsiSI fragment that was ligated into pAAVS1-TLR (opened with AsiSI and AscI), resulting into pAAVS1-TLR6, serving as AAVS1 targeting vector with the TLR6 reporter insert. pU6Rosa-CAG-Cas9 for expression of Rosa26 sgRNA and Streptococcus pyogenes (Sp) Cas9 was constructed by ligation of the DNA oligonucleotides sgRosa-A and sgRosa-B into the BbsI sites downstream of a human U6 promoter into plasmid pU6(BbsI). The U6-sgRosa cassette was recovered as AscI fragment and inserted into pCAG-Cas9-bpA-EF1-BFP, upstream of the CAG promoter driving SpCas9 expression, followed by a BFP coding region under control of the human EF1α promoter. Plasmid pTLR-donor was generated by whole plasmid PCR amplification using 5′-phosphorylated primers TLRtv-1 and TLRtv-2 and pTLR-repair (Addgene ID 64322)  as template, followed by DpnI digestion of the template and religation of the PCR fragment. The modification of pTLR-repair removes the Start codon of the Venus coding region, eliminating background fluorescence upon transient transfection. For construction of pTLR-donor-tetO, pTLR-donor was opened with SalI and ligated with a 448 bp SalI fragment from plasmid pTREtightbi, a derivative of pTREtight (Clontech) that contains seven Tet operator elements. For construction of plasmid pTLR-donor-UAS, pTLR-donor was opened with SalI and ligated with two tandem copies of a 151 bp PCR fragment amplified with primers UAS-for/UAS-rev, each containing 5 UAS elements, using plasmid pUAS-luc2 (Addgene ID 24343) as template. Plasmid pCAG-Rad18UBD was constructed by generation of a 225 bp PCR fragment, including the UBD domain of human Rad18 between codon 191–240, amplified from plasmid myc-hRad18 (Addgene ID 68827) as template using primers Rad18-for and -rev. The PCR product was incubated with DpnI to remove the plasmid template, digested with PacI and MluI and ligated into the backbone of plasmid pCAG-Rad51-bpA-EF1-BFP opened with Pac and MluI. Plasmid pU6Rosa-CAG-Rad18 for expression of Rad18 was constructed by amplification of a 1536 bp PCR product, including the full length human Rad18 coding region, using primers Rad18-for2 and -rev2 and Addgene plasmid 68,827 as template. The PCR product was incubated with DpnI, digested with PacI and MluI and ligated into the PacI-MluI sites of plasmid pU6Rosa-CAG-Rad51-bpA-EF1BFP, replacing the Rad51 coding region. Plasmid pCAG-RNF169UBD for expression of the RNF169UBD was constructed by amplification of a 219 bp PCR fragment, including the UBD domain of human RNF169 between codon 662–709, using plasmid pcDNA5-FRT/TO-Flag-RNF169 (Addgene ID 74243) as template with primers RNF169-for2 and -rev. The PCR product was incubated with DpnI, digested with PacI and MluI and ligated into the backbone of plasmid pCAG-Rad51-bpA-EF1-BFP opened with Pac and MluI. Plasmid pCAG-RNF169 for expression of the full length human RNF169 was constructed by ligation of a 2167 bp PCR product amplified with primers RNF169-for and –rev (using a synthetic gene as template) into the backbone of plasmid pCAG-Rad51-bpA-EF1-BFP opened with Pac and MluI. Plasmid pCAG-BRCA1 for expression of BRCA1 was constructed by amplification of 5640 bp PCR fragment, including the full length human BRCA1 coding region, using plasmid pDEST-FRT/T0-GFP-BRCA1 (Addgene ID 71116) as template and primers BRCA1-for and -rev. The PCR product was incubated with DpnI, digested with PacI and MluI and ligated into the backbone of plasmid pCAG-Rad51-bpA-EF1-BFP opened with Pac and MluI. Plasmid pCAG-i53 was constructed by amplification of a 273 bp PCR fragment, including the i53 coding region, using plasmid pcDNA3-Flag-UbvG08 (Addgene ID 74939) as template and primers i51-for and -rev. The PCR product was incubated with DpnI, digested with PacI and MluI and ligated into the backbone of plasmid pCAG-Rad51-bpA-EF1-BFP opened with Pac and MluI. Plasmids pCAG-BRCA1-, pCAG-TetR-, and pCAG-Gal4-Rad18UBD for expression of Rad18UBD fusion proteins were constructed by amplification of specific PCR products that were digested with PacI and PvuI and ligated into the PacI site of pCAG-Rad18UBD. BRCA1 was amplified as 5681 bp PCR product using primers BRCA1-for and -rev2 and pCAG-BRCA1 as template, The TetR coding region was amplified as 1445 bp PCR fragment using primers TetRsc4-for and –rev and plasmid pU6MS2Rosa-CAG-MS2ditetRsc as template. pU6MS2Rosa-CAG-MS2ditetRsc was generated by cloning of a 739 bp PCR fragment amplified with primers scTetR-for2 and rev2 from pU6MS2Rosa-CAG-MS2ditetR into the PmlI site of pU6MS2Rosa-CAG-MS2ditetR to duplicate the TetR coding region, separated by 5 copies of a (GGGGS) linker, to generate a single chain Tet repressor coding region as described. We used a single chain TetR that combines two TetR monomers separated by a linker sequence  to enable the binding of TetR fusion proteins as a single protein. The Gal4 DNA binding domain was amplified as 561 bp PCR fragment from pActPL-Gal4DBD (Addgene ID 15304) using primers Gal4-for and –rev. The same PCR products were ligated into the PacI site of pCAG-RNF169UBD to generate the plasmids pCAG-BRCA1-RNF169UBD, pCAG-TetR-RNF169UBD and pCAG-Gal4-RNF169UBD for the expression of RNF169UBD fusion proteins. The AAVS1 targeting vector for Cas9 expression pAAVSI-NEOwt-CAG-Cas9v3a-bpA was generated by isolation of a 4.7 kb PacI-XhoI fragment isolated from pCAG-Cas9v3a-bpA followed by ligation into the AflII and SalI site of pAAVS1-NEOwt CAG-tetR-donor U6 acceptor, a derivative of Addgene plasmid 60,431. PX458_GABPA_2 and PX458_CREB1_1 were a gift from Eric Mendenhall & Richard M. Myers (Addgene plasmid # 64255 and 64,939). For cloning of pU6LMNB1-CAG-Cas9 the DNA oligonucleotides sgLMNB1-A and sgLMNB1-B (target sequence: GGGGTCGCAGTCGCCATGGC) were annealed and ligated into the BbsI sites downstream of a human U6 promoter into plasmid pU6(BbsI) chimaeric RNA. The U6-sgRNA fragment was then PCR amplified and cloned into the AscI site of CAG-Cas9-EF1-BFP. The same approach was used to clone sgRNA against LMNA (target sequence: TGGGACGGGGTCTCCATGGC) and AAVS1 (target sequence: GGGGCCACTAGGGACAGGAT) using oligonucleotide pairs sgLMNA-A/−B and sgAAVS1-A/−B. To construct the targeting vector pTetO-CREB1-3xFLAG a 1.5 kb fragment containing the CREB1 homology arms (HA) flanking 3X FLAG Tag insert was synthesized (Thermo Fischer Scientific). This fragment was then cloned via SgfI/ SpeI sites into the plasmid backbone of pTLR-donor-tetO. The same strategy was used to derive pTetO-GABPA-3xFLAG (2 kb HA), pTetO-AAVS1-3xFLAG (1.6 kb HA) and pTetO-LMNA-3xFLAG (1.1 kb HA) that were synthesized and cloned into the SgfI/SpeI sites of pTLR-donor-tetO. To clone pTetO7-LMNB1-GFP, the 448 bp SalI fragment was isolated from pTLR-donor-tetO and ligated upon end filling into the SnaBI site of AICSDP-10:LMNB1-mEGFP (Addgene #87422). All coding regions and functional elements of the constructed plasmids were confirmed by Sanger sequencing. The plasmids will be distributed via the Addgene repository (www.addgene.org).
Generation of reporter cell lines
The human IPS cell lines BCRT and JWT (a kind gift of Harald Stachelscheid, BIH, Berlin) were used to generate TLR-Cas9 transgenic cell lines. These lines were grown in feeder-free conditions in Essential 8 (E8) or E8 Flex complete media (Thermofischer Scientific, #A1517001) on Vitronectin (Life Technologies, #A14700) coating in a 6-well cell culture plate. Plasmid DNA (pbs-U6-sgAAVS1-T2 (Addgene ID 41818), pAAVS1-TLR  and pAAVS1-NEOwt-CAG-Cas9v3a-bpA vector were diluted at a concentration of 0.5 μg/μl in deionized water. The AAVS1-TLR and -Cas9 Knockin vectors harbor a Puromycin and Neomycin (G418) antibiotic resistance marker respectively for the selection of targeted clones. Passaging was performed using PBS-EDTA dissociation buffer in fresh media containing Y27632 selective ROCK Inhibitor (Tebu-Bio, #21910–2301-2). The cells were transfected using Lipofectamine 3000 transfection reagent (Life Technologies, #L3000001) using 1 μg plasmid each for sgRNA, Cas9 and TLR vectors following manufacturer’s protocol. Puromycin (Sigma-Aldrich #P8833-25MG) (0.5 μg/ml) selection was started 48 h after transfection and continued until single colonies were formed. Puromycin resistant single clones were picked and then selected by using G418 (ThermoFisher #11811064) at the concentration of 100 μg/ml for 10 days. After the antibiotic selection, 24 colonies were expanded for genotyping. Genomic DNA was isolated using Wizard genomic DNA purification kit (Promega #A1125). Genotyping PCRs were performed for knock-in of TLR construct (puro 5′) and Cas9 (Neo 5′) as well as AAVS1 WT locus specific as a control. The PCR reaction for TLR Knockin was performed using primers ST_puro_gt_fw and ST_puro_gt_rv, Phusion HF DNA polymerase (NEB # M0530 L) and 200 ng genomic DNA using following conditions; 98 °C for 3 min, 35 cycles of 98 °C 30 s, 58 °C 30 s and 72 °C for 90 s. Cas9 KI was confirmed by Neo 5′ KI PCR using primers ST_neo_gt_fw and ST_neo_gt_rv using conditions at 98 °C for 5 min, followed by 35 cycles of 98 °C 30 s, 61 °C 30s, 72 °C for 90 s. AAVS1 WT PCR was performed using primers hAAVS1-For and hAAVS1-Rev by amplifying at 98 °C for 5 min, followed by 40 cycles of 98 °C 30 s, 60 °C 30 s, 72 °C for 45 s. After confirmation of TLR and Cas9 insertion by genotyping, selected clones were tested for activity of the TLR allele by FACS-based assay and one clone was chosen for further assays. The expression of Cas9 and of pluripotency markers in TLR/Cas9 reporter cells was confirmed by immunofluorescence staining.
HEK293 cells were maintained in Dulbecco’s Modified Eagle’s medium with Glutamax (Gibco) supplied with 10% fetal bovine serum (Gibco). The HEKTLR6 line was generated by using pAAVS1-TLR6, the same AAVS1 targeting vector as described above for the TLR construct with the difference that the TLR6 insert can be used for both plasmid and ssODN-based repair. The transfections for generation of the HEKTLR6 line were performed using sgRNA for AAVS1 and Cas9 plasmids (750 ng each) using Xtreme-gene transfection reagent (Roche #6366244001). Antibiotic selection was performed using 0.4 μg/ml Puromycin. Single clones were generated and genotyped in the same way as described above for the iPS TLR/Cas9 lines. All cell lines were confirmed for the absence of mycoplasma using the PCR assay of Uphoff and Drexler .
Transfection of cells
TLR/Cas9 transgenic iPS cells were passaged one day prior to transfection using PBS-EDTA dissociation buffer. Plasmid vectors sgRNA-Rosa3, TLR donor template and one or a combination of Rad18UBD or RNF169UBD fusion vectors (750 ng each) were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s protocol. HEKTLR6 cells were seeded in 24-well plates (50,000 cells per well) one day before transfection. Cells were transfected with pU6Rosa-CAG-Cas9, pTLR-donor-tetO or pTLR-donor-UAS template and one or two plasmids for the expression of Rad18UBD or RNF169UBD fusion proteins (375 ng each) using Xtreme gene transfection reagent (Merck) according to manufacturer’s protocol. All samples were transfected in triplicate wells. For the targeting of endogenous genes in HEK293, cells were seeded in 48-well plates (50,000 cells per well) one day before transfection. The transfection of HEK293 cells were performed using sgRNA for the targeted locus, Cas9, targeting vector and/or the fusion proteins (360 ng each plasmid per well) using Xtreme-gene transfection reagent.
Analysis of HDR and NHEJ by flow cytometry
FACS analysis for TLR reporter assays was performed upon 72 h after transfection. For preparation of cells for FACS analysis the medium was aspirated from wells and each well was washed with PBS. Accutase (Thermo Fisher Scientific, #A1110501) was used to detach iPS cells while Trypsin was used for the HEK cells. The cells were collected after adding appropriate media by centrifugation at 300 g for 4 min and finally resuspended in 500 μl PBS. The cells were kept on ice and FACS analysis was performed immediately. Single cells were gated for BFP positive populations and the frequency of Venus (HDR) and RFP (NHEJ) positive cells was determined using a BD LSR Fortessa flow cytometer (BD Biosciences). The analysis of the entire population (without BFP gating) leads to the same proportion of Venus and RFP positive cells but lower absolute numbers. The results from triplicate wells of each sample were used to calculate the mean value and standard deviation. For the FACS analysis of LMNB1-GFP Knockin in HEK cells, the medium was aspirated from wells and each well was washed with PBS. The cells were then harvested using Trypsin and collected down after adding appropriate media by centrifugation at 300 g for 4 min and finally resuspended in 500 μl PBS. The cells were kept on ice and FACS analysis was performed immediately. For LMNB1-GFP assay single cells were gated for the frequency of GFP positive cells reporting for HDR using BD LSR Fortessa flow cytometer (BD Biosciences). Flow cytometry settings were controlled by using untransfected cells or cells transfected with a BFP or Venus expression vector as shown in Figure S7.
For the TLR locus the frequency of NHEJ and HDR was determined by Illumina amplicon sequencing via Genewiz (Amplicon EZ; GENEWIZ Germany GmbH). For this purpose, DNA was isolated from pooled triplicates of the same experiment used for FACS (Fig. 4). An outer PCR reaction (591 bp) was performed using primers located outside the homology arms, while a second PCR reaction was performed using primers amplifying a shorter PCR product of 245 bp. For the first PCR reaction primers CAG2 and Venus-rev were used alongwith Optitaq DNA polymerase (Roboklon, # E2600–02) and 200 ng DNA per reaction using these conditions; initial denaturation at 95 °C for 1 min, followed by 35 cycles of 95 °C 20 s, 61 °C 35 s and 72 °C for 40 s and final extension at 72 °C for 5 min. For the second PCR amplification, Venus-for-Ilumina and Venus-rev-illumina Optitaq DNA polymerase and 20 ng of purified 1st PCR product following these conditions; initial denaturation at 95 °C for 2 min, followed by 35 cycles of 95 °C 20s, 61 °C 25 s, 72 °C for 40 s and final extension 72 °C for 5 min.
For the LMNA, GABPA, CREB1 and AAVS1 loci, the frequency of NHEJ and HDR was determined by Illumina amplicon sequencing via Genewiz (Amplicon EZ; GENEWIZ Germany GmbH). DNA was isolated from each well and a first PCR was performed using primers with at least one primer binding site locating outside the homology arm (Table S1). For the CREBP1 and GABPA gene, the PCR reaction was performed using hCREBP1 5HDR-F + hCREBP1-3HA intern-R and hGABPA 5HA intern-F + hGABPA 3-HR-R primers, LongAmp DNA polymerase (Neb #M0323S) and 200 ng DNA per reaction using these conditions; initial denaturation at 94 °C for 30 s, followed by 35 cycles of 94 °C 15 s, 59 °C 40 s and 65 °C for 1 min 30 s. Final extension was done at 65 °C for 10 min. For the AAVS1 and LMNA locus, the PCR reaction was performed using HR-AAVS1-F + AAVS1-R and LMNA-outer-F + LMNA-outer-R primers, LongAmp DNA polymerase (Neb #M0323S) and 200 ng DNA per reaction using these conditions; initial denaturation at 94 °C for 30 s, followed by 35 cycles of 94 °C 15 s, 61 °C 40 s and 65 °C for 1 min 30 s/2 min (AAVS1/LMNA). Final extension was done at 65 °C for 10 min. The second PCR amplification was performed using following primers (Primer LMNA 3′ inner fw_illumina, Primer LMNA 3′ inner rev_illumina, hGABPA_ intFw_illumina, hGABPA_ intRev_illumina, hCREB1_IntFw_Illumina, hCREBP1_intRev_Illumina, AAVS1 inner fw_illumina, and AAVS1 inner rev_illumina), Q5 DNA polymerase (Neb #M0491S) and 50 ng of gelextracted 1st PCR product with these conditions; initial denaturation at 98 °C for 30 s, followed by 35 cycles of 98 °C 10 s, 62 °C 30 s, 72 °C for 15 s and final extension at 72 °C for 2 min. The PCR products were purified on column using the GeneJET PCR Purification Kit (Thermo Scientific, #K0702). 2 μg of each purified PCR reaction was submitted for deep sequencing. The analysis of deep sequencing data was performed using Crispresso  and Crispresso2 (http://crispresso.pinellolab.partners.org/) online bioinformatics tools designed for analysis of Crispr-based gene editing data. Additionally, a custom Amplicon-sequencing analysis pipeline (licensed from Bioinformatics. Expert UG, Berlin, Germany) was used for data analysis which needs the rawdata fastq-files for the paired reads as well as the reference sequence provided as fasta-file. The main functionalities of that pipeline were implemented in R statistics (version: 3.6.0). The alignment algorithm was performed using the R function “pairwiseAlignment()” from the R package Biostrings (version: 2.52.0). In a first step, the paired reads were aggregated and cleaned to reduce potential technical sequencing errors. This was done for each pair of reads as following: First, the R2-read was converted to its reverse complement sequence, the quality score was reversed. Second, the R1- and R2-read were aligned locally to identify the overlap between the read pairs. The local alignment was performed with very high penalty values for gap opening and gap extension to avoid an alignment with gaps, which might shift the bases during aggregation. Third, R1 and the reverse complement R2-sequence (rcR2) were stitched together at the aligned sequence. If bases differ between R1 and rcR2, the base was chosen, which had the highest quality score on that position. Missing bases at the end were filled with “N”. Fourth, the 15 bases from the 5′ and 3′ ends of the reference sequence were aligned locally to the aggregated sequence to identify the sequence boundaries. In this step, two mismatches or two Insertions/Deletions were allowed. Afterwards, all bases in front or after the alignment were chopped. So, a final aggregated sequence was created for each read, which starts and ends with the boundary bases of the reference sequence. In the second part of the pipeline, all aggregated reads were then aligned globally to the reference sequence (alignment reward/penalty scores: match = 1, mismatch = 0, gapOpen = − 2, gapExtension = 0). The alignment scores were chosen to favor long gaps over small gaps and mismatches. Finally, all unique aggregated sequences were sorted and counted.
For all experiments, data are shown as mean ± standard deviation. Statistical significance was determined as indicated in each results part. A P value of less than 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, USA).
Synthetic oligonucleotides used in this study were purchased from Eurofins Genomics (Ebersberg, Germany) and are shown in Table S1.