Cell culture
We used a iCRISPR-Cas9 line derived from 409-B2 human induced pluripotent stem cells13 (GMO permit AZ 54-8452/26), HEK293 cells (ECACC, 85120602) grown in DMEM/F-12 (Gibco, 31330-038) supplemented with 10% fetal bovine serum (FBS) (SIGMA, F2442) and 1x NEAA (SIGMA, M7145); K562 cells (ECACC, 89121407) grown in Iscove’s modified Dulbecco’s media (ThermoFisher, 12440053) supplemented with 10% FBS; Jurkat cells (ATCC, TIB-152) grown in RPMI 1640 medium (Gibco, 21875034) supplemented with 10% FBS; Caco-2 cells (DSMZ, ACC169) grown in MEM Eagle (SIGMA, M4655) supplemented with 20% FBS and 1× NEAA; SNL 76/7 (ECACC, 07032801) grown in KnockOut DMEM (Gibco, 10829018) supplemented with 2 mM GlutaMAX (Gibco, 35050061) and 10% FBS; as well as CHO-K1 cells (ATCC, CCL61) grown in Ham’s F-12K medium (Gibco, 21127022) supplemented with 10% FBS. All cell lines were authenticated by the supplier via certificate of analysis and additionally in-house by checking morphology. All cell lines were tested negative for mycoplasma contamination before and after the experiments. 409-B2 hiPSCs were grown on Matrigel Matrix (Corning, 35248) in mTeSR1 medium (StemCell Technologies, 05851) with supplement (StemCell Technologies, 05852) and MycoZap Plus-CL (Lonza, VZA-2011) that was replaced daily. Medium for other cells was replaced every second day. At ~80% confluency, adherent cells were dissociated using EDTA (VWR, 437012 C) and split 1:6 to 1:10 in medium supplemented with 10 µM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Calbiochem, 688000) for one day after replating. K562 and Jurkat cells were split by 1:6 to 1:10 dilution after 1 week. Cells were grown at 37 °C in a humidified incubator with 5% CO2. For experiments in which Cas9 should be produced by 409-B2 iCRISPR hiPSCs 2 µg/ml doxycycline (Clontech, 631311) was added to the media three days before transfection. For generation of single cell-derived cellular colonies (Fig. 3b), cells were dissociated and thoroughly separated using TrypLE Express (ThermoFisher, 12605036) and seeded in different dilutions.
Production of sgRNA
Secondary structure prediction of gRNAs was done using the RNAstructure web server23. Chemically synthesized crRNAs and tracrRNAs as well as oligonucleotides for sgRNA production were from IDT (Coralville, USA) (Supplementary Data). For the production of sgRNAs by in vitro transcription (IVT) ssDNA templates were designed to contain the T7 promoter sequence in front of the sgRNA coding sequence. These ssDNA templates were hybridized with short complementary ssDNA for 2 min at 95 °C, cooled for 10 min at 20 °C to form a dsDNA T7 promoter and used for IVT according to the manufacturer’s protocol (T7 High Yield RNA synthesis kit NEB, E2040S). The IVT reaction mix was incubated overnight at 37 °C before 2 µL of TURBO DNase (Invitrogen, AM2238) was added and incubation continued for 30 additional minutes. IVT products were purified with the MEGAclear Transcription clean-up kit (Invitrogen, AM1908). For experiments in which sgRNAs are expressed in the cell, dsDNA with a U6 promoter sequence in front of the sgRNA coding sequence were designed and amplified (98 °C 30 s; 35 × [98 °C 10 s, 61 °C 20 s, 72 °C 25 s]; 72 °C 5 min) using Phusion HF MasterMix (Thermo Scientific, F-531L). Amplifications were analyzed using EX agarose gels (Invitrogen, G4010-11) and PCR products were purified using solid phase reversible immobilization (SPRI) beads28.
Production of modified plasmids
pSpCas9(BB)-2A-GFP (PX458) (Addgene #48138) and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene #62988) were used as vectors to integrate sequences coding for full-length gRNAs carrying either the normal gRNA backbone or the locked gRNA backbone (Supplementary Data) using BbsI (ThermoScientific, FD1014) restriction digest and cloning as described by the depositor. Cloned plasmids were used for heat-shock transformation of E.coli strain Stbl3 (Invitrogen, C737303). Bacteria were streaked on ampicillin (100 µg/ml) containing LB-agar (SIGMA, L2897) plates and incubated overnight at 37 °C, yielding single colony density. Single colonies were used to inoculate overnight cultures in LB-medium (Sigma, L3022) followed by plasmid DNA extraction with the Plasmid Plus Midi Sample Kit (Qiagen, 12941). To exclude unintended plasmid mutations that differ between plasmids modified to express the gRNAs with either the normal or locked backbone we used aliquots of purified plasmids for plasmid library preparation and sequencing. First, plasmids were sonicated three times with a Bioruptor (Diogenode), with the output selector switched to (H)igh to yield fragments of ~0.15 to 0.8 kb. The sheared fragments were blunted for 30 min at 20 °C with the Quick Blunting Kit (New Engand Biolabs, E1201L), purified with SPRI beads28, adapter-ligated for 30 min at 20 °C with the Quick Ligation Kit (New England Biolabs, M2200L), again purified with SPRI beads, double-indexed by PCR-amplification and finally purified with SPRI beads. After Illumina sequencing, we compared the mapped sequences of the modified plasmids with the respective plasmid reference sequences using the integrative genomics viewer (IGV)29.
Lipofection of oligonucleotides
Lipofection (reverse transfection) was done using the alt-CRISPR manufacturer’s protocol (IDT) with a final concentration of 7.5 nM of each gRNA that had been previously formed by hybridization of crRNA and tracrRNA. In brief, 0.75 µl RNAiMAX (Invitrogen, 13778075) and the respective gRNAs were separately diluted in 25 µl OPTI-MEM (Gibco, 1985-062) each and incubated at 20 °C for 5 min. Both dilutions were mixed to yield 50 µl of OPTI-MEM including RNAiMAX and gRNAs. The lipofection mix was incubated for 20–30 min at 20 °C. 409-B2 iCRISPR hiPSCs were dissociated using EDTA for 5 min and counted using the Countess Automated Cell Counter (Invitrogen). The lipofection mix, 100 µl containing 25,000 dissociated cells in mTeSR1 supplemented with Y-27632 and 2 µg/ml doxycycline were thoroughly mixed and transferred to 1 well of a 96-well plate covered with Matrigel Matrix (Corning, 35248). Media was changed to regular mTeSR1 media after 24 h.
Electroporation of oligonucleotides and RNPs
Electroporation of oligonucleotides was done using the B-16 program of the Nucleofector 2b Device (Lonza) in cuvettes for 100 µl Human Stem Cell nucleofection buffer (Lonza, VVPH-5022), containing 1 million cells, 78 pmol electroporation enhancer, and 320 pmol of gRNA. For RNP based editing either 252 pmol S.p. HiFi Cas9 (IDT) or 252 pmol S.p. Cas9 (IDT) were used. For generation of ‘worst-target’ inserted cells 200 pmol of single-stranded DNA donor (Supplementary Data) was additionally added to the nucleofection buffer. When plasmids px458 or px459 were used for editing 5 µg of the respective plasmids was added to the nucleofection buffer. No doxycycline was added to the media of cells that were used for RNP or plasmid-based editing. For generation of HDR-edited cells, the small molecule M3814 (MedChemExpress, HY-101570) was added to the media for two days post-electroporation to increase HDR efficiency30.
Illumina library preparation and sequencing
At least five days after transfection cells were detached using TrypLE (ThermoFisher, 12605036), pelleted, and resuspended in 15 µl QuickExtract (Lucigen, QE0905T). Incubation at 65 °C for 10 min, 68 °C for 5 min, and 98 °C for 5 min was performed to yield single stranded DNA. Primers containing adapter overhangs for Illumina sequencing (Supplementary Data) were used to amplify each locus in a T100 Thermal Cycler (Bio-Rad) using the KAPA2G Robust PCR Kit (SIGMA, KK5024) with buffer B and 3 µl of cell extract in a total volume of 25 µl. The thermal cycling profile of the PCR was: 95 °C 3 min; 34× (95 °C 15 s, 65 °C 15 s, 72 °C 15 s); 72 °C 60 s. P5 and P7 Illumina adapters with sample specific indices were added in a subsequent PCR reaction31 using Phusion HF MasterMix (Thermo Scientific, F-531L) and 0.3 µl of the first PCR product. The thermal cycling profile of the second PCR was: 98 °C 30 s; 25× (98 °C 10 s, 58 °C 10 s, 72 °C 20 s); 72 °C 5 min. Amplifications were verified by size separating agarose gel electrophoresis using 2% EX gels (Invitrogen, G4010-11). The indexed amplicons were purified using solid phase reversible immobilization (SPRI) beads28. Double-indexed libraries were sequenced on a MiSeq (Illumina) yielding 2 ×150 bp (+7 bp index). After base calling using Bustard (Illumina) adapters were trimmed using leeHom32.
Amplicon sequence analysis
Bam-files were demultiplexed and converted into fastq files using SAMtools33. CRISPResso34 was used to analyze fastq files for percentage of wild type and indel sequences. For analysis of precise insertion frequency of ‘worst-case’ targets in single cell-derived cellular clones, the expected amplicon was provided to CRISPResso to call the HDR event. Analysis was restricted to amplicons with a minimum of 70% similarity to the wild type sequence and to a window of 20 bp from each gRNA. Unexpected substitutions were ignored as putative sequencing errors.
In vitro binding and cleavage assay
The in vitro cleavage of target DNA was adapted from Zetsche et al.35. In brief, a ribonucleoprotein (RNP) mix consisting of 340 nM gRNA (hybridized crRNA/tracrRNA) and 165 nM Cas9 or dead (d)Cas9 (both IDT) was incubated for 20 min at 20 °C, and subsequently mixed with 75 nM of the corresponding dsDNA targets, that had been previously generated by hybridizing complementary ssDNA (Supplementary Data) for 2 min at 95 °C followed by 10 min at 20 °C. Samples were incubated at 37 °C for 16 h and then subjected to Proteinase K or RNaseA (in combination, alone or none of each). In case of both treatments, Proteinase K was done prior to RNase A digest. Proteinase K (NEB P8107S, 800 U/ml) digest was carried out at 50 °C for 30 min and RNaseA (Qiagen 1007885, 2 mg/ml) digest was performed at 37 °C for 30 min. Samples were subjected to size separating agarose gel electrophoresis using 4% EX gels (Invitrogen, G401004) for qualitative analysis as well as to capillary gel electrophoresis with a High Sensitivity DNA chip (Agilent, 5067-4626) for quantitative analysis of cleaved and uncleaved, as well as bound and unbound, target DNA.
Statistics and reproducibility
Bar graphs in figures were plotted and SEM error bars were calculated using GraphPad Prism 6 software. The number of replicates is stated in the respective figure legends. No statistical method was used to predetermine sample size. The experiments were not randomized. Samples were prepared unblinded but in parallel. Analysis was performed based on numerical sample names, without the identity of the samples known during the analysis.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
