Cloning and plasmids
RFP transposon PB512-B for random insertion monitoring was purchased from System Biosciences Inc. hyPB vector was obtained from Wellcome Trust Sanger Institute (pCMV_hyPBase)11. Plasmid vector pCRTM-Blunt II-TOPO® was from Invitrogen and Cas9, nCas9 and SP-dCas9-VPR were obtained from Addgene (Addgene plasmid #41815, #41816, #63798). Finally, SB100X and pT4-HB were a kind gift from Dr. Zsuzsana Zizsvak. gRNAs were produced using The Zero Blunt TOPO PCR cloning kit (Invitrogen). with a gblock gene fragment (Integrated DNA Technologies) containing U6 promoter, 20 nt target site, gRNA scaffold and terminator. gRNA-TRAC and gRNAs for CasX, CjCas9, LbCpf1 and SaCas9 were designed and validated in the lab, gRNA AAVS1-3 sequence was previously described23,24. Off targets of both gRNAs were computationally predicted with cas-offinder25 and cutting frequency determination was calculated using Doench, Fusi et al.24 scoring model (Supplementary Fig. 8).
Nuclease, nickase and dead Cas9 fusions to hyPB and ½ emGFP transposon were performed by Golden Gate assembly using BspQI enzyme and standard methods. CasX, CjCas9, LbCpf1, and SaCas9 fused to hyPB R372A_K375A_D450N expressing vectors were cloned on pcDNA4 using Golden Gate assembly and Esp3I according to manufacturer recommendations.
MC plasmid of ½ emGFP SMN1 transposon was obtained amplifying it from previously described ½ GFP transposon and cloning into pMC BESPx MCS1 (Systems Biosciences) and transformed into YCY10P3S2T Minicircle Production Strain (Systems Biosciences). MC production was performed according to the manufacturer’s protocol.
Different mutations were introduced into hyPB sequence fused to Cas9 (Cas9_PB plasmid) by site directed mutagenesis following QuikChange Lightning mutagenesis kit’s instructions (Agilent). Primers were designed with QuickChange Primer Design to achieve following mutations to the hyPB sequence: M194V, R245A, G325A, R372A, K375A, R376A, E377A, E380A, D450N, S564P. Cas9-hyPB_R372A_K375A_D450N coding plasmid was deposited at Addgene (#179381). All plasmids are available upon request. PB ½ emGFP SMN1 was obtained by introducing the first half of emGFP sequence and SMN1 intron 6 sequence into PB acceptor vector. pT4 SMN1 2/2 emGFP was obtained by adding a second half SMN1 intron 6 and partial emGFP in SB100X transposon vector. emGFP sequences containing SMN1 were obtained from DYP004reporter26, a kind gift from Sri Kosuri.
Luciferase transposon was obtained by cloning firefly luciferase preceded by a CMV promoter into pMC BESPx MCS1.
Transposon and HDR templates of different sizes were generated by cloning a partial cDNA (NC_000006.12) fragment upstream of the split emGFP reporter system.
Lentiviral payload was prepared from pSICO obtained from Addgene (Addgene plasmid #11578) and Cas9 and Esp3I cloning sites were introduced to provide a Golden Gate acceptor vector for the PB variants combinatorial library.
Cell culture, transfection and electroporation
Hek293T cell line (ATCC CRL-3216), C2C12 cell line (ATCC CRL-1772) and K-562 cell line (gifted by Dr. Meyerhans; ATCC CRL-3343) were cultured at 37 °C in a 5% CO2 incubator with Dulbecco’s modified Eagle medium, supplemented with high glucose (Gibco, Thermo Fisher), 10% fetal bovine serum, 2 mM glutamine and 100 U penicillin/0.1 mg/ml streptomycin. Cell lines were purchased with an authentication report prior purchase. Hek293T cell’s transfection experiments were performed using lipofectamine 3000 reagent following the manufacturer’s instructions or polyethyleneimine (PEI, Thermo Fisher Scientific) at 1:3 DNA-PEI ratio in OptiMem. Cells were seeded the day before to achieve 70% confluency on transfection day (usually 290,000 cells in adherent p12 well plate). C2C12 and K-562 cells electroporation experiments were carried out by using SE Cell Line 4D-Nucleofector and SF Cell Line 4D-Nucleofector kits (Lonza), respectively, and using the manufacturer’s instructions for 100 µl single Nucleocuvette on the 4D-Nucleofector (Lonza). Plasmid molar ratio was 1 transposase:2.5 gRNA:2.5 transposon or 1 Cas9:2.5 gRNA:2.5 HDR template using either 0.076 pmol FiCAT or Cas9 for p12 well plate.
emGFP splicing based reconstitution assay
Hek293T cell line containing pT4 SMN1 2/2 emGFP was generated by PEI mediated transfection of SB100X and pT4 SMN1 2/2 emGFP DNA constructs, followed by single clone expansion and PCR genotyping (Supplementary Table 5). A positive clone was selected and expanded and used for subsequent assays.
For emGFP reconstitution assay, FiCAT, gRNA and transposon plasmids were transfected in a 1 FiCAT:2.5 gRNA:2.5 transposon ratio using 0.076 pmol FiCAT or hyPB and 0,19 pmol transposon and gRNA for a 12 wells plate. For the MC transposon, a molecular ratio of 1 FiCAT:2.5 gRNA:5 MC-transposon showed better results. On-target insertion was measured 5 days post transfection by emGFP fluorescence. Off-target insertion was measured 15 days post transfection of RFP transposon by RFP fluorescence and calculated as the subtraction of % GFP fluorescence (on-target) to % RFP fluorescence (overall insertion). emGFP and RFP expression measured at (BD LSRFortessa; BD Biosciences. Blue 488 nm laser with 530/30 filter and Yellow Green 561 nm laser with 610/20 filter) (Supplementary Fig. 17). BD FACSDiva version 6.2 and version 8.0.2 for analysis.
Junction PCRs for insertion site sequencing
Junction PCR was performed on sorted cells with BD FACSAria (Biosciences). Selected cells had on-target insertion of PB ½ emGFP or RFP transposon targeting AAVS1, TRAC, lama 271.1, rosa26 target site on reporter cell line, Hek293T, K-562, c2c12 or liver tissue. In the case of liver tissues a second nested PCR was performed. Genomic DNA was extracted using DNeasy Blood and tissue kit (Qiagen). Primers were designed by the 3′ ITR of the transposon (forward) and targeting the different genomic locations studied taking into account insertion at + or − strand (reverse) (Supplementary Table 5).
Library prep and Illumina sequencing for targeted insertion analysis
We implemented STAT-PCR18 amplifying the 3′ ITR of the transposon DNA coupled to Illumina sequencing to capture genome integration sites with high sensitivity. Genomic DNA was extracted from enriched cells by flow cytometry sorting using DNeasy Blood and tissue kit (Qiagen) and fragmented to 500 bp fragments using Q800R3 Sonicator. End repair, A-tailing, and ligation of Y-adapter were performed using KAPA Hyper Prep Kit (KR0961–v5.16) and 3 μg of fragmented genomic DNA, followed by AMPure XP SPRI bead purification at 1X ratio. After adapter ligation, each sample was split in two and amplified with GSP5′ or GSP3′ to capture 5′ and 3′ junctions, respectively. To capture 5′ and 3′ transposon-genome junctions, two nested PCRs were performed using KAPA HiFi DNA Polymerase following manufacturer protocol: PCR1 with P5_1 and PB_5_GSP1 or PB_3_GSP1 in a 25 μl final volume and PCR2 with P5_2 PB_5_GSP2 or PB_3_GSP2 in a 25 μl final volume. 5′ and 3′ PCR products were purified with AMPure XP SPRI bead purification at 1X ratio, mixed in equimolar ratio and sequenced with Illumina Miseq Reagent Kit V2–500 cycles (2 × 250 bp paired end). Three microliters of 100 μM custom primers index 1 and read 2 were added to the sequencing reaction.
Bioinformatics analysis of targeted integration analysis
Illumina reads were clustered with usearch v11.0.66727 and mapped to the reference using bwa-mem v0.7.1728. For on-target insertion characterization, reads covering 5′ and 3′ junctions from the target insertion site were selected with Python scripting and Samtools 1.1029. Number of indels was obtained with CRISPR-GA30. For on-target and off-target experiments, clustered reads that mapped against the vector were selected and mapped against the reference genome using bwa-mem in short reads and minimap2 v2.1731 in long reads. Significance of the insertion peaks was assessed with macs2 v2.2.532 algorithm and taking into account the standard deviations of read start and end positions. We estimated the LOD of the method by diluting the positive UMIs computationally, we selected randomly 1%, 10%, 25%, 50% and 99% of the positive UMIs while maintaining the 100% negative UMIsand repeated the dilution process for 100 replicates . We analyzed the dilution samples with the previously described pipeline and applied a logarithmic transformation to the fold enrichment of on-target peaks (the predictor variable). Then, we extrapolated the dilution at fold enrichment 0 in order to determine the minimum percentage of on-target sample needed to detect a significant peak (Supplementary Fig. 10). We estimated between 0.1% and 9% of LOD for all positive samples.
in vivo targeted insertion to mice liver
Animal experimentation procedures were approved by the Animal Experimentation Ethic Committee of Barcelona Biomedical Research Park. C57BL/6J, 8–10 weeks old, were used for this study. Animals were purchased from Jackson Laboratories, male and female were used without distinction. FiCAT mRNA was produced by in vitro transcription with RiboMAX Large Scale RNA Production Systems-T7 (Promega) following the manufacturer’s instructions. Rosa26 gRNA33 was purchased from Synthego. FiCAT mRNA or plasmid, sgRNA or gRNA plasmid targeting Rosa26 and PB512-B, luciferase or GFP MC transposon were injected via retro-orbital using two delivery methods. For in vivo JetPEI delivery plasmids were used in a 1 FiCAT:2.5 gRNA:2.5 transposon molecular ratio. A total of 60 μg of nucleic acids was complexed with In vivo JetPEI (Polyplus transfection) at NP ratio 7. For hydrodynamic injection, a total of 10 to 10.2 μg of nucleic acids were used (6 μg MC-luciferase transposon/MC-GFP transposon, 2 μg FiCAT pDNA/3 μg FiCAT mRNA, 2 μg gRNA pDNA/1.2 μg sgRNA targeting Rosa26.2).
Nucleic acids were diluted with PBS and 7% of animal body weight in ml was injected in less than 7 s via retro-orbital systemic injection.
Whole body imaging of luciferase expression was performed at different timepoints after FiCAT-gRNA-transposon or transposon control administration with IVIS spectrum imaging system (Caliper Life Sciences). Images were taken 5 min after intraperitoneal injection of D-Luciferin potassium salt (Gold Biotechnology) according to the manufacturer’s instructions.
For qPCR copy number analysis of PB512-B transposon, animals were euthanized 10 days after injection and the liver was isolated and homogenized. Genomic DNA was extracted from liver samples with DNeasy Blood and tissue kit (Qiagen) Transposon relative Copy number to Tfrc endogenous gene was obtained by qPCR (primers listed in Supplementary Table 5).
PB combinatorial library screening
DNA library was produced by Twist Bioscience, cloned into a lentiviral vector containing Cas9 and Esp3I golden Gate cloning site, and transformed into ElectroMax Stbl4 competent cells (Thermo Fisher), ensuring 100 times representation of each combinatorial variant. Plasmids were purified with HiPure Maxiprep kit (Life technologies) and cotransfected with envelope and packaging plasmids into Hek293T cells to produce lentivirus. Lentivirus was harvested, filtered and titered comparing functional titer (GFP fluorescent cells by GFP carrier lentivirus infection) with qPCR based titer34. Reporter cell line containing C-t half of GFP sequence was infected at MOI 1 corrected by PB copy number (to avoid bias for cloning efficiencies between cycles). Infected cells were transfected with ½ GFP plasmid and gRNA targeting AAVS1 sequence into the reporter target side, transfections were performed as previously described. On-target positive cells were selected by flow cytometry sorting 5 days after transfection and genomic DNA was extracted. Genomic DNA product was used to be cloned and start a new cycle, PB was amplified by PCR from genomic DNA and cloned into a lentiviral vector containing Cas9 with Golden Gate assembly.
PB structural modeling
A 3D structure of the Trichoplusia ni PB transposase protein was obtained by Robetta Web protein structure prediction server (http://robetta.bakerlab.org). The core domain (131–550aa) was predicted by Rosetta Comparative Modeling method that is based on Monte Carlo algorithm with embedded Cartesian-space minimization and all-atom optimization35. The tertiary structure fold was analyzed and validated with SPServer and ProSa-Web knowledge-based methods (Supplementary Fig. 3). Secondary structure was analyzed with PSIPRED and HHPred machine-learning based methods. PB’s core was then modeled for refinements with PyMOL by comparative protein modeling methods. The refinement process was guided by the superimposition of the PB model with cryo-EM HIV-1 strand transfer complex intasome (PDB ID: 5U1C) consisting of the HIV integrase tetramer bound to viral DNA and target host DNA and X-ray diffraction Tn5 transposase complex structure (PDB ID: 1MUS36). Strand-transferring DNA and donor DNA were extrapolated from the superimpositions of HIV-1 intasome and Tn5, respectively. The nucleotides in the interface in contact with the protein were analyzed with X3DNA as double-strand DNA. We used statistical potentials to score the interaction between protein and DNA and generate a theoretical PWM37. The theoretic PWM is obtained by testing all potential double-strand DNA sequences in the interface, ranking them with the statistical potentials and selecting the top to make a multiple sequence alignment. During the submission of this manuscript a cryo-EM structure became available, which shows important agreement with modeling performed17. Cryo-EM structure of PB transposase strand transfer complex (PDB ID: 6X67) confirmed the general fold of the model and the domains we hypothesized were responsible for the contact with donor and target DNA.
Statistics and reproducibility
No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
