MATERIALS AND METHODSOligonucleotidesAll oligonucleotides are ordered from Merck KGaA, Darmstadt, Germany. The sequences for all oligonucleotides used can be found in Supplementary Table S1 and S2.Cell cultureTissue cultures of human embryonic kidney 239T (HEK293T) cells and normal immortalized human fibroblasts (HMF, clone MJ2646, a gift from Dr Bin Liu, Danish Cancer Society, Denmark) were cultured in Dulbecco's modified Eagle's medium (DMEM) (LONZA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% GlutaMAX (Gibco), and penicillin/streptomycin (100 units penicillin and 0.1 mg streptomycin/ml) in a 37°C incubator with 5% CO2 atmosphere and maximum humidity. HEK293T and MJ2646 cells were passaged typically every 2–3 days at 1:8 and 1:3 ratio, respectively, when reaching approximately 90% confluence. This was done by washing cells twice (equal volume as growth medium) gently with phosphate buffered saline (PBS, 1×) without calcium and magnesium, followed by cell detachment by 0.05% Trypsin-EDTA for 3–5 min at 37°C.Resource of plasmidsThe following vectors were used: lentiCRISPRv2 (a gift from Feng Zhang, Addgene plasmid # 52961) and pUC19 (gift from Joachim Messing, Addgene plasmid # 50005). The pMAX-GFP (VDF-1012) was supplied by the Amaxa nucleofection kit. The PiggyBac hybase plasmid is generously provided by Prof. Jacob Giehm Mikkelsen from the Department of Biomedicine, Aarhus University (http://www.giehmlab.dk). Alternatively, the PiggyBac transposase expression vector can be purchased from SBI system Biosciences (Cat. # PB210PA-1).Generation of eccDNA biosensorsThe ECC biosensor fragments were synthesized by Gene Universal Inc. (229A Lake Dr Newark De 19702, USA) and validated by Sanger sequencing. The ECC biosensor fragments, which contains fluorophore gene markers of enhanced green fluorescent protein (EGFP) and the monomeric derivative of DsRed fluorescent protein (mCherry), were sub-cloned into a PiggyBac transposon vector (from Prof. George M. Church's group, http://arep.med.harvard.edu) carrying the pac gene, encoding a puromycin N-acetyl-transferase that confer resistance to puromycin (see Supplementary Figures S1 and S2). The final PiggyBac transposon-based ECC biosensor plasmids (CAG-ECC and TRE-ECC) were verified by restriction enzyme digestion.Generation of CRISPR/Cas9 gRNAsAll CRISPR gRNAs were designed with the online software tool CRISPOR v4.2. The gRNAs were selected selecting based on (i) minimal off-targets, i.e. off-targets that typically requires more than three mismatches for perfect annealing elsewhere; (ii) predicted activity over 30% provided by the CRISPOR web tool; (iii) avoid strong secondary structure of the guide sequences and (iv) absent of poly-thymine (max 3) and the BsmBI restriction site in the guide. All CRISPR gRNA oligos with the corresponding overhangs used for Golden Gate Assembly were ordered from Merck KGaA, Darmstadt, Germany (Supplementary Table S1).To generate CRISPR gRNA expression vector, we used a previous optimized protocol. Briefly, for each gRNA, two complementary guide oligonucleotides (100 pmol each) in 1× NEB buffer 2 (in a total volume of 20 μl) were first denatured at 95°C for 5 min, using a heating block, followed by slow annealing by turning off the heating block. For Golden Gate Assembly, the following reaction mix was prepared: 1 μl of the above annealed oligonucleotides, 100 ng of the lentiCRISPRv2 plasmid, 1 μl FastDigest BsmBI restriction enzyme (Thermo Fisher Scientific), 1 μl T4 Fast ligase (Thermo Scientific), and 2 μl T4 ligase buffer in a total volume of 20 μl. Golden Gate Assembly was performed in a thermal cycler using the following program: Ten cycles of 37°C for 5 min and 22°C for 10 min; one cycle of 37°C for 30 min; and one cycle of 75°C for 15 min. The ligation product was stored at 4°C or used directly (2 μl ligation product) to transform competent Escherichia coli cells. Using this protocol, we have experienced that over 95% of the bacterial clones are positive by PCR screening using a U6 forward primer (5′-GAGGGCCTATTTCCCATGATTC-3′) and the antisense guide oligonucleotide (template strand of the gRNA spacer). Sanger sequencing validated all gRNA vectors used in this study.Cell transfectionAll transfections in this study were conducted with the X-tremeGENE 9 transfection reagent (Roche) in 24-well plate, if not stated elsewhere. Briefly, 60,000 cells, counted with nucleocounter NC-100 (Chemometec), were typically seeded into a 24-well plate one day before transfection. For each transfection, a total of 250 ng plasmid DNA and 0.75 μl X-tremeGENE 9 transfection reagent (1:3 ratio of μg total DNA relative to transfection reagent), were mixed in 50 μl OptiMEM (Gibco) and incubated at room temperature for 15 min. The transfection mixture was homogeneously added to the cells. Unless stated elsewhere, for transfection of pairs of CRISPR plasmid, equal amount of each CRISPR plasmid was used. As transfection control, pUC19 or GFP plasmid was used for control groups. For evaluation of endogenous eccDNA formation by CRISPR pairs, 1.5 × 106 HEK293T cells or 2.2 × 106 HMF cells were seeded in 10-cm cell culture dish and transfected next day at ∼50% cell confluence.Generation of ECC biosensor stable clonesStable ECC biosensor clones were generated by transfection of 60,000 HEK293T cells with 200 ng ECC biosensor plasmid and 50 ng PiggyBac hybase plasmid using X-tremeGENE 9. Twenty four hours post transfection, transfected cells were cultured in complete medium supplemented with puromycin (1 μg/ml). One-week after transfection, the puromycin resistant cells were trypsinized, single cell was manually picked under a stereomicroscope and cultured in 96-well until 70–80% confluence. Single clones normally evolve after 14 days in culture. The stable clones were further expanded and the copy number of the integrated ECC biosensor was assessed by Southern blot, probing for EGFP, using the ECC biosensor plasmid as reference in 1, 5 and 10 copies/genome.Fluorescence-activated cell sorting (FACS)Cells, dissociated with 100 μl trypsin-EDTA, were suspended in 100 μl 5% FBS–PBS and transferred to a 96 deep-well plate on ice. Cells were spun down ∼800 × g (2000 rpm) for 5 min and the supernatant was removed by gently inverting the plate. For fixation, 200 μl 4% paraformaldehyde was added to the cells and mixed gently by pipetting. After fixing for 15 min at room temperature, the fixed cells were washed twice with 5% FBS–PBS. Lastly, cell pellets were re-suspended in 500 μl PBS and cells were kept at 4°C until FACS analysis. FACS was performed using a BD LSRFortessa (supported by the FACS CORE facility, Department of Biomedicine, Aarhus University) with at least 30 000 events collected for each sample. Data were analyzed using Flowjo software.Time-course analysis of ECC biosensorThe CAG-ECC (clone #1–9) and TRE-ECC (clone #1–15) were transfected with Cr1 and Cr2. 72 hours after transfection (defined as passage 1), the transfected cells were dissociated with 100 μl trypsin-EDTA (37°C for 5 min) and re-suspended in 200 μl 5% FBS-PBS medium. One-third of the cells were analyzed by FACS, one-third of the cells were used for PCR analysis, and the remaining one-third of the cells were passaged to a new 24-well plate. The cells were passaged every second day at a one-third ratio until passage 6.EccDNA purification in human cellsCells were harvested typically at 48 h after transfection or harvested from a fraction of the transfected cells in time-course experiments at various passages. Suspended, trypsinized cells were pelleted at 425 x g (2000 rpm), 5 min. The supernatant was removed and cells were lysed for eccDNA analysis (see the following optimization method); either by genotyping, using outward PCR and Sanger sequencing, or by Southern blot.Optimization of endogenous eccDNA purificationTo assess the methods for endogenous eccDNA purification, three different protocols were tested before outward PCR: (i) genomic DNA column purification; (ii) cell lysate; (iii) plasmid DNA column purification. For each of the three methods, cells were counted and fractionated in three different concentrations: 105 cells, 104 cells and 102 cells. Then, to each cell sample was spiked in 105 copies of pUC19 plasmid as internal control and protocols (i); (ii) or (iii) were executed.Genomic DNA column purification Genomic DNA was purified according to protocol (#056-60, A&A Biotechnology). In brief, cells were lysed by protease for 30 min at 55°C, the RNA was removed by RNase A/T1 mix (2 μg/sample, Thermo) and genomic DNA was washed and purified by gravity flow on an ion exchange membrane (AX tissue), followed by DNA precipitating at 14,000 x g for 30 min at 2°C, 70% ethanol wash and finally dissolving the DNA pellet in 40 μl Tris–Cl 10 mM, pH 8.0. Of the total 40 μl DNA solution, 32 μl was transferred for linear DNA removal, saving the remaining 8 μl at −20°C until PCR analysis.Cell lysate Cells were suspended in 0.2 ml lysis buffer (KCl 50 mM, MgCl2 1.5 mM, 0.5% NP40 and 0.5% Tween 20, 10 mM Tris pH 8.5) and incubated with 10 μl proteinase K (19.1 mg/ml, Thermo) for 2.5 h at 55°C. Proteinase K was heat-inactivated at 95°C for 10 min, cooled down at room temperature for 15 min, and 32 μl DNA solution was transferred for linear DNA removal, storing the rest of the cell lysate at –20°C until PCR analysis.Plasmid DNA column purification Plasmid DNA from cells was purified according to protocol (#010-50, A&A Biotechnology) after completed cell lysis in 0.6 ml L1 suspension buffer with 30 μl proteinase K (19.1 mg/ml, Thermo) for 6 h at 55°C, and removal of RNA by RNase A/T1 mix (2 μg/sample, Thermo) at room temperature for 10 min. Plasmid DNA was washed and purified by gravity flow on an ion exchange membrane (Plasmid 10 AX), followed by DNA precipitating at 14 000 x g for 30 min at 2°C, 70% ethanol wash and finally dissolving the DNA pellet in 40 μl Tris–Cl 10 mM, pH 8.0. Of the total 40 μl DNA solution, 32 μl was continued for linear DNA removal, saving the remaining DNA at –20°C until PCR analysis.Removal of linear DNAA maximum of 4–5 μg total DNA (most cases < 2 μg) was digested with four fast digest units (Thermo Scientific), using an endonuclease that did not cleave investigated eccDNA, e.g. for most experiments XbaI (endogenous eccDNA) or EcoRI (ECC biosensor) was used. Endonuclease digestion was completed after 1 hour in a 40 μl volume at 37°C, followed by heat-inactivation, according to enzyme protocol (e.g. 65°C, 20 min). To the total 40 μl endonuclease digested DNA was added a 35 μl Plasmid-Safe DNase mixture (Epicentre, Illumina), containing 1.5 μl Plasmid-Safe DNase (15 units), 3 μl ATP (25 mM), 7.5 μl 10× DNase reaction buffer and 23 μl H2O. Samples were incubated in a heating block at 37°C for 16 h. Next day, linear DNA digestion was continued by adding 5 μl fresh Plasmid-Safe DNase mixture every two hours, consisting of 1.5 μl Plasmid-Safe DNase (15 units), 3 μl ATP (25 mM ATP) and 0.5 μl 10× DNase reaction buffer and when reaching 90 units and ∼24 h digestion, DNase was heat-inactivated at 70°C for 30 min (see Supplementary Figures S13 and S14). In case of the cell lysate protocol, the quick protocol included max 4 μg DNA, endonuclease digestion of a 40 μl cell lysate volume, conducted directly after cell lysis in 1× fast digest buffer at 37°C for 16 h, in combination with 10 units Plasmid-Safe DNase and 2 μl ATP (25 mM). After heat-inactivation of the endonuclease, the Plasmid-Safe DNase treatment was continued for additional 24–48 h (dependent on DNA input), adding fresh Plasmid-Safe DNase mixture step-wise and reaching up to 100 units/sample.Polymerase chain reaction (PCR)All PCR reactions were performed with the high-fidelity platinum Pfx polymerase in the presence of 2× enhancer solution (#11708013, Thermo). All PCR primers were ordered from from Merck KGaA, Darmstadt, Germany (Supplementary Table S2).Southern blot analysisSouthern blot was performed using an optimized method from our group. In brief, a total 15–25 μg genomic DNA was digested with EcoRI restriction enzyme overnight. The enzyme digested DNA was separated on 1% agarose gel for 16 h at 30 V. The DNA was transferred on to a membrane by vacuum blotting. PCR primers for generating the probe: 5′-ATTCACCGTCTCATCCAGCGAGGGCGATGCCACCTAC-3′ and 5′-TAAGTCGGTCTCATCAGTGGTTGTCGGGCAGCAGCAC-3′ (500 bp EGFP probe). Probe labelling was performed using the Prime-It II Random Primer Labeling Kit according to the manufacturer's instructions. Pre-hybridization and hybridization steps were carried out at 42°C. Excess probe was washed from the membrane with SSC buffer, and the hybridization pattern was visualized on X-ray film by autoradiography. A detailed protocol with step-by-step guidance can be found at our group page: www.dream.au.dk.KaryotypingCells were incubated with Chromosome Resolution Additive, diluted 1:10 000, and 500 ng/ml cholcemide for 2.5 h at 37°C. The culture was harvested by incubating with TrypLE for 10–20 min at 37°C. Cells were collected by centrifugation and the pellet was re-suspended in 0.4% KCl, gently vortexed and subsequently incubated for 30 min at 37°C. After re-centrifugation, the pellet was re-suspended by adding fixative drop-wise up to 2 ml using 1 volume acidic acid and 3 volume methanol. After 20 min fixation at room temperature, cells were spun down and the fixation step was repeated twice. Chromosome spreads were prepared by dripping the cell-suspension onto wet glass slides in a humid atmosphere at 37°C. The slide glasses were stained, using quinacrine and mounted in antifade solution.Fluorescent in situ hybridization (FISH) of telomere and whole chromosome 18 paintingSlides were made with each of the cells suspensions prepared for Karyotyping. Hybridization with PNA-telomere probe in red was performed, followed by DAPI as counterstain. Automated scanning of the slides was applied to identify and locate metaphases, and image identified metaphases were captured using the automated scanning software. The telomere probe was stripped and re-hybridization with whole chromosome 18 probes in green followed by DAPI as counterstain. Relocation of previously captured metaphases followed by another capture of painted metaphases was carried out. For wildtype parental cells, we analyzed 65 metaphases. For CRISPR transfected cells, we analyzed 113 metaphases. A more detailed FISH protocol can be acquired from the authors.Sanger sequencingAll Sanger sequencing in this study were conducted using the Mix2Seq Kit in Eurofins Genomics (Munich, Germany). Sanger sequencing results were analyzed with SnapGene Viewer or four peaks software.Deep sequencingPurified PCR products were mixed with End Repair Mix (BGI Tech.), incubated at 20°C for 30 min, then purified with QIAquick PCR purification kit (Cat No./ID: 28104). Purified PCR products were mixed with an A-Tailing reaction (BGI Tech.), incubated at 37°C for 30 min and purified with QIAquick PCR purification kit. PCR-free adaptor ligation was carried out by mixing the above 3′ adenylate DNA, PCR Free index adapters (BGI Tech.), T4 DNA Ligase and buffer. The reaction was incubated at 16°C for 16 h and purified with QIAquick PCR purification kit. The ligated PCR products were purified by gel-electrophoresis (2% agarose) and the QIAquick Gel Extraction Kit (Qiagen). The molecular length of the final sequencing library was determined with Agilent 2100 bioanalyzer (Agilent DNA 1000 Reagents) and sequenced with HiSeq2500 System (HiSeq SBS Kit V4, Illumina).Analysis of amplicon sequencing resultsThe theoretical circular sequences of dsCRPcircle 1q23.2:0.57 kb and TRIM28circle exon 1-2 were generated based on human hg38. Clean reads were aligned against the theoretical reference using the BWA-mem algorithm. Duplicate removing was not suitable for amplicon sequencing and thus omitted. Reads across the theoretical end joining site (junction) were used for indel-calling. Soft-clip reads were omitted.StatisticsAnalysis of correlation was conducted with Pearson rank test.
The eccDNA biosensor vectors and CRISPR-C plasmids are available at Addgene repository (https://www.addgene.org/YonglunLuo/). The eccDNA biosensor reporter cells can be requested from Yonglun Luo directly. All detail protocols can be found atwww.dream.au.dkor requesting to the corresponding author Yonglun Luo.