Abstract
Precise gene editing and genome engineering by CRISPR technology requires simultaneous delivery of multiple DNA-encoded components into living cells rapidly exceeding the cargo capacity of currently utilized viral vector systems. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck. We implement hybrid DNA techniques (MultiMate) for rapid and error-free assembly of currently up to 25 functional DNA modules in a single baculoviral vector enabling CRISPR-based genome engineering. Utilizing homology-independent targeted integration (HITI), we achieve up to 30% correct genome interventions in human cells, including precision docking of large DNA payloads in the ACTB locus. We demonstrate baculovirus-vectored delivery of prime-editing toolkits for seamless DNA search-and-replace interventions achieving, with a single vector, highly efficient cleavage-free trinucleotide insertion in the HEK3 locus without any detectable indels. Our approach thus unlocks a wide range of editing and engineering applications in human cell genomes.
Main
CRISPR/Cas represents a game-changing gene editing tool1. A programmable DNA nuclease (Cas9) is precisely guided to a specific DNA locus by means of a short single guide RNA (sgRNA) to elicit double strand DNA breaks (DSBs) subsequently repaired through non-homologous end-joining (NHEJ) introducing small insertions-deletions (indels), giving rise to frameshift mutations and functional gene knock-outs (KOs)1. Unpredictable indels that likewise occur are however undesirable in the context of therapeutic gene editing. Precise gene editing in contrast is typically achieved through homology directed repair (HDR) by providing a DNA template flanked by homology arms of variable length1,2, resulting in precise gene correction or knock-in (KIs)1. HDR activity however is intrinsically low and mostly restricted to S/G2 cell cycle phases3-5, reducing the efficiency of the desired gene editing outcome. Significant effort is being made to increase the efficacy of CRISPR-HDR by means of small-molecule NHEJ inhibitors6, cell cycle stabilised Cas9 variants7,8 and other strategies9,10, however, gene editing efficiency in vivo has remained low.
More recently, homology-independent targeted integration (HITI), was shown to efficiently induce base pair precise KIs in both dividing and non-dividing cells11-13 by exploiting NHEJ and Cas9 cleaved donors, with exciting potential for gene editing applications in vivo11,14. Moreover, single base substitutions can also be achieved by using base editors (BEs) involving catalytically impaired Cas9 variants fused to cytosine or adenine deaminase 15,16and, more recently, prime editors (PEs) using Cas9 nickase fused to reverse transcriptase, achieving genomic interventions with little to no indels and reducing the risks associated with DSBs17. BE, PE and HITI share in common a reliance on multiple functional DNA and protein elements that must be simultaneously delivered into target cells. This limits their applicability, in particular for future bona fide therapeutic interventions that necessitate a systemic approach and where co-transfection of plasmids and proteins, and likewise co-infection of viral vectors, will be problematic or unfeasible. In summary, the large DNA cargo capacity required for implementing these next-generation genomic interventions in vivo stands at odds with the limited cargo capacity of available technology, including the currently dominating adeno-associated virus (AAVs) and lentivirus (LVs) vector systems18. In marked contrast, baculoviral vectors (BVs) have a heterologous DNA cargo capacity far exceeding AAV and LV18-20. BVs, suitably pseudotyped to alter host cell tropism, are already widely used to transduce mammalian cells and living organisms19-22. We demonstrated in a proof of concept that BV is suitable for CRISPR-HDR mediated small (∼0.7 kb) DNA insertions with low efficacy (∼5%)19,23, as would be expected for HDR3-5.
Here we unlock baculovirus as a vector of choice for next-generation genome intervention approaches, fully exploiting its unprecedented DNA cargo capacity and versatility. We implement and deploy state-of-the-art DNA assembly technology (MultiMate) for error-free bottom-up assembly of multifunctional DNA circuitry comprising the DNA cargo to be inserted as well as all the DNA encoding all CRISPR modalities required to achieve highly efficient genome intervention in a baculovirus-vectored approach.
Results
We had previously developed methods to rapidly assemble functional DNA elements into multicomponent circuitry in baculoviral vectors24-26. Here, we optimized and fine-tuned our approach by incorporating time-tested MultiSite Gateway recombination modalities to assemble with ease currently up to 25 distinct DNA elements of various sizes (Fig.1a, Extended Data Fig.1a-e, Supplementary Methods), importantly aiming to significantly reduce prokaryotic elements carried over into the baculovirus which can compromise vector integrity during manufacturing27 (Extended Data Fig.1f). We validated the MultiMate system by expressing the eight subunits of human chaperonin CCT/TRiC complex28 in insect cells from a BV comprising MultiMate assembled DNA (23 kb) (Extended Data Fig.1g-j). We then assessed MultiMate to efficiently deliver large multicomponent DNAs for expression and live cells imaging of up to 7 fluorescently labelled proteins (MultiMate-Rainbow) in human cells (Fig.1b, Extended Data Fig.2a). MultiMate assembly yielded remarkably low error rates validating our approach (Extended Data Fig.2b). We deployed BVs harbouring 18 and 23.4 kb of MultiMate assembled functional DNA to efficiently transduce HEK293T, HeLa, H4 and SH-SY5Y leading to homogeneous expression and correct subcellular localization of all fluorescently labelled proteins (Fig.1b, Extended Data Fig.2c). Moreover, we created MultiMate-CellCycle (9.1 kb) as an improved cell cycle tracking tool implementing FUCCI reporters29 as well as H2B-iRFP expression for accurate transduction efficiency monitoring and cell cycle stage assessment (Fig.2c,Extended Data Fig.2d,e). MultiMate-CellCycle BVs efficiently transduced HEK293T, HeLa, H4 and SH-SY5Y cells highlighting differences in their respective cell cycle progressions (Extended Data Fig.2f) and enabling 12-hrs time lapse imaging on living HeLa cells, allowing cell cycle tracking also during FUCCI-unstained stages (M-G1 transition) (Fig1.c,Supplementary Video 1). These results comprehensively validate the MultiMate assembly platform enabling a wide range of baculovirus-vectored applications.
Baculovirus-vectored homology independent targeted integration (HITI)
To date, baculovirus-vectored gene editing approaches were confined to CRISPR-HDR of small insert DNAs with low efficacy19,22,23. HITI toolkits using a viral vector required donor and Cas9/sgRNA to be split between two AAVs due to their limited cargo capacity11, restricting successful gene editing to the fraction of co-infected cells. Moreover, manufacturing complete ‘all-in-one’ HITI vectors is not possible when viral packaging is performed in mammalian cells (typically HEK293T for AAV, LV), because simultaneous expression of Cas9 and sgRNA would inevitably excise the HITI donor, fatally compromising virus production. In marked contrast, BVs are manufactured in insect cells, where the mammalian promoters controlling Cas9 and sgRNA expression are silent and all-in-one HITI construct packaging into the BV is thus entirely feasible.
To minimise unpredictable indels and maximise correctly-edited alleles, we sought to analyse and compare HDR and HITI-2c11 strategies by targeting the intronic β-actin (ACTB) locus, introducing a synthetic C-terminal exon fused to mCherry and a self-cleaving peptide (T2A)30 followed by a puromycin selection cassette (Fig.2a). We eliminated any possibility of leaky expression by outfitting our DNAs with the Cas9 inhibitor AcrII431 under control of a dual prokaryotic and baculoviral promoter (J23119-polH). An additional module expressing eGFP under the constitutive CMV promoter was added to track transduction efficiency. Despite comparable transduction of HEK293T cells, Multimate-HITI-2c resulted in a 3- to 4-fold higher percentage of mCherry+ cells compared to Multimate-HDR (Fig.2b-c, Extended Data Fig.3h). The BV backbone was rapidly diluted as we expected, while mCherry+ cells were stably maintained over time (Fig.2b-c) with absolute gene editing efficiencies reaching 5% (HDR) and 20% (HITI-2c) (Fig.2c). Notably, when compared to plasmid transfection, baculovirus-vectored delivery increased gene editing efficiency up to 4-fold regardless of the approach (Extended Data Fig.3a-c). To confirm correct gene editing, BV transduced cells were selected with puromycin and then expanded in the absence of selective pressure, demonstrating stably maintained mCherry expression in close to all (>98%) cells (Fig.2d). Successful editing was confirmed by PCR genotyping (Extended Data Fig.3e), the expected mCherry subcellular localization (Fig.2e,Extended Data Fig.3f) and the predicted ACTB::mCherry molecular weight (68 kDa) in western blot (Fig.2f, Extended Data Fig.3g). MultiMate-HITI-2c ACTB outperformed HDR editing in all BV transduced cell lines (Extended Data Fig.3h). For optimal efficacy, we prepared VSV-G pseudotyped BVs19 and transduced HEK293T, HeLa, H4 and SH-SY5Y at different multiplicities of transduction (MOT) achieving higher transduction (up to 100%) and editing efficiencies (up to 30%) depending on the cell line (Fig.2h, Extended Data Fig.3i). To our best knowledge this is the first assembly and delivery platform to enable efficient homology independent targeted integration in mammalian cells using a single all-in-one viral vector.
Safe-harbour integration of large DNA payloads
Precision docking of large multicomponent DNA circuitry in mammalian genomes is a prerequisite for bona fide genome engineering, but remains an impeding challenge for currently available viral delivery systems which are constrained by their intrinsic packaging limitations (AAV: ∼4 kb; LV: ∼8 kb)18,20. We assessed the aptitude of our system for precision DNA docking exploiting MultiMate-HITI-2c (Supplementary Methods). We used a new Cre insertion site to generate a series of HITI-2c payloads ranging from 4.7 kb to 18 kb with mTagBFP as a transduction efficiency reporter, resulting in all-in-one MultiMate plasmids of up to 30 kb (Fig.3a). Transduction with EMBacY BV32 markedly outcompeted plasmid transfection and editing in HEK293T (Extended Data Fig.4a-d). Upon puromycin selection, cells remained >98% mCherry+ (Extended Data Fig.4e) confirming precise 5’-end integration. We observed silencing of the 3’-end fluorescent marker correlated with cargo size, which we could fully restore by hygromycin selection (Fig.3b, Extended Data Fig.4f). We confirmed correct integration by PCR genotyping and Sanger sequencing (Fig.3c,d). We deployed MultiMate-HITI-2c 18K-CGH (30 kb) for baculovirus-vectored delivery with VSV-G pseudotyped BV achieving 100% transduction efficiency in both HEK293T and SH-SY5Y giving rise to 20% and 30% absolute genomic insertion efficiency, respectively (Fig.3e). Of note, mCherry expression remained constant over time in the absence of any selective pressure while silencing of 3’ eGFP expression (Fig.3f,g) was again promptly restored by puromycin/hygromycin selection, remaining stable thereafter (Fig.3h). Our results demonstrate that safe-harbour integration of extensive DNA payloads with base-pair precision can be achieved with high-efficiency using all-in-one MultiMate-HITI-2c BVs, setting the stage for future large synthetic gene regulatory network engineering in human genomes.
Highly efficient baculovirus-vectored search-and-replace gene editing
Base editors (BEs)15,16 and prime editing (PEs)17 are new additions to the CRISPR toolkit that could potentially correct up to 89% of the human disease-causing mutations in the absence of DNA cleavage17. PE in particular, can be harnessed to precisely edit genomes with little to no indels production. PE exploits the nickase Cas9-H840A fused to reverse transcriptase from MMLV (PE2) to make a ssDNA copy of the engineered PegRNA at the edited site, allowing for the generation of all possible point mutations, insertions (up to 44 bp) and deletions (up to 80 bp) in the absence of DNA cleavage17. We chose here to insert a CTT trinucleotide in the HEK3 locus using the prime editing enzyme PE217. PE2 coding sequence spans 6.3 kb (excluding promoter) encoding for a 240 kDa protein that previously could be virally delivered only through multiple split-intein lentiviral vectors17 due to cargo limitation, but is entirely within the packaging size of BV. We therefore assembled MultiMate-PE2 comprising PE2, HEK3 PegRNA cassette, VSV-G (for pseudotyping), aeBlue chromoprotein33 (for visual readout of virus titer) and mTagBFP (for transduction tracking) (Fig4. a, Extended Data Fig.5a). MultiMate-PE2 plasmid transfection resulted in 13% CTT insertion in HEK3 as assessed by Sanger sequencing and deconvolution using ICE34 (Extended Data Fig.5b,c). MultiMate-PE2 BVs production could be easily monitored (Extended Data Fig.5d) with excellent transduction efficiencies in the cell lines tested (Fig4b,d and Extended Data Fig.5e). Sanger sequencing on HEK3 locus amplicons from unsorted cells showed correct CTT editing with base-pair precision (Fig.4c) with correct editing contributions after deconvolution of 15-45%, depending on the cell line, and undetectable indels (Fig.4e). We increased the transduction titer and observed a dose-dependent effect boosting correct editing events (up to 45%) again without any indels detected (Extended Data Fig.5f-i) unlocking PE to our baculovirus-vectored approach for future, conceivably multiplexed applications including therapeutic interventions.
Discussion
The limited packaging capacity of currently dominating viral vectors (AAV, LV) markedly constrains precise genome editing interventions18,20. We demonstrated here the efficacy of our baculovirus-vectored approach to overcome this limitation. We developed a rapid error-free DNA assembly (MultiMate) to facilitate vector construction with a view to improve BV manufacturing. We implemented homology independent targeted integration (HITI) for precise DNA insertion achieving efficient C-terminal tagging in the ACTB locus, markedly outcompeting HDR efficiency. Using our approach, we demonstrated precision safe-harbour integration with outstanding efficacy of large multicomponent DNA payloads. There is no indication that we reached the cargo limit of baculovirus-vectored delivery. In fact, given the wide variation in size of naturally occurring baculoviruses35 we expect that delivery of DNA cargos exceeding 100kb will likely be feasible, enabling insertion of entire metabolic pathways and gene regulatory networks in safe-harbour sites or elsewhere in genomes, additionally incorporating DNA insulator elements36,37 to support sustained gene expression. Importantly, we demonstrate the utility of our approach for seamless search-and-replace gene editing by implementing recently developed prime editing (PE) technology17. Particularly PE, is considered safer when compared to HDR or HITI-2c, which both rely on DNA cleavage. Using a single baculovirus vector, we achieved PE-mediated trinucleotide insertion (CTT) in the HEK3 locus with up to 45% efficiency in the absence of detectable indels. Taken together, our results establish baculovirus as a vector of choice for precision engineering of large DNA cargoes in human genomes, and we anticipate baculovirus-vectored large-scale genome interventions, even combining safe-harbour integration with concomitant, if needed multiplexed, base or prime editing strategies, enabling complex synthetic biology and therapeutic approaches in the future.
Methods
Gibson assembly of DNA elements
An extensive list of constructs maps and assembly strategies is provided in Supplementary Table 1. ENTR and DEST vectors were generated using Gibson assembly (NEB Builder Hi-Fi DNA assembly #E2621S) following manufacturer’s instructions. Fragments were mixed with a backbone to insert ratio of 1:1 (>3 fragments), 1:2 (<2 fragments), 1:5 (fragments <300 bp) in 5 µl total volume and supplemented with 5 ul 2xNEB Builder Hi-Fi mix followed by incubation at 50°C for 1 hour. 2 ul of the assembly mix were transformed into homemade electrocompetent Top10 or Pir+ E.Coli. followed by recovery at 37°C while shaking for 1 hour and plating on LB/agar plates with the appropriate antibiotics. All the precursors vectors generated in this study were assembled using Gibson assembly of synthetic DNA fragments, oligonucleotides, digested vectors or PCRs amplified with Herculase II fusion (Agilent# 600675). A portion of pENTR-D-TOPO (Invitrogen) was used as backbone for all the pMMK ENTR vectors. attL/R sites on pMMK ENTR vectors were obtained by overlap extent PCRs of long oligonucelotides. attR1-Ccdb-Chlo-attR2 cassettes for generating pMMACE DEST were PCR amplified from pInducer-2038. pMMDS DEST was generated by replacing the Chloramphenicol cassette with OriColE1 PCR amplified from pACEMam119. H2B-iRFP was PCR amplified from pCAG-H2BtdiRFP-IP39, EYFP-Tubulin and mTFP1-Actin were amplified from 5-colours MultiBac vectors19. pMDK 7 kb and 12 kb were generated by cloning promoterless and ATG-less portions of SMG1(NM_015092.5) CDS. Mito-mCherry was generated by fusing mCherry from 7TGC40 to the COX8 mitochondrial targeting sequence from 5-colours MultiBac vectors19. CyOFP141 fused to the endoplasmic reticulum targeting sequence (ER) was synthesised by Twist Bioscience. mAG β-catenin was obtained through Gibson assembly of PCR amplified mAG from pL-EF1a mAG-hGeminin42, and β-catenin from pL-EF1a β-catenin SV40 Puro43. Golgi targeting sequence (GTS) mTaBFP was synthesised by Twist Bioscience. polH or p10 cassettes were amplified from MultiBac vectors32,44, CCT isoforms were PCR amplified from synthetic vectors from GenScript. SpCas9 was PCR amplified from px4591, sgRNAs cloning was performed by overlap extent PCRs of hU6 and scaffold fragments amplified from px4591. HDR and HITI-2c templates were amplified by Gibson assembly of genomic DNA fragments, mCherry was amplified from 7TGC40, T2A Puro from px4591. sgRNAs target in the HITI-2c donors were included as overlapping ends between fragments. polH/J23119 AcrII431 was synthesised by Twist Bioscience. In the HITI-2c payloads for safe-harbour integration, mCherry T2A Puro was replaced with T2A mCherry P2A Puro. P2A was generated by overlap extent PCR of long oligonucleotides. The loxP site was PCR amplified from pACEBac132,44, Hygromycin, IRES and eGFP were PCR-amplified from p1494 vectors45. PE2 was obtained by restriction digestion from pCMV PE217 and the HEK3 PegRNA was PCR amplified from pU6-Sp-pegRNA-HEK3_CTT_ins17. aeBlue33 was synthesised by Twist Bioscience and VSVG-G was amplified from pMD2.G (Didier Trono lab, Addgene plasmid # 12259).
LR recombination
LR recombination was carried out using LR Clonase II (Thermo Fisher #11791020) or LR Clonase II plus (Thermo Fisher #12538120). Although LR Clonase II plus is specifically designed for MultiSite Gateway recombination, LR Clonase II worked with comparable efficiency in our hands. LR reactions were carried out following manufacturer’s instructions. One DEST and four ENTR vectors were diluted to 20 femtomoles/µl each in TE buffer pH 8.0 (Thermo Fisher #12090015). 1 ul of each diluted vector was added to a 0.2 ml PCR tube with 2 ul LR Clonase II and 3 ul TE buffer, followed by a brief spin and incubation at 25°C for 16 hours. The next day the reaction was terminated by addition of 1 ul proteinase K (provided with LR Clonase II enzymes) and incubation at 37°C for 10 minutes. 2-3 ul were transformed into homemade electrocompetent Top10 or Pir+ E.Coli, followed by 2 hours recovery at 37°C and plating on LB/agar plates with the appropriate antibiotics. LR recombination products were predicted using APE46 with custom recombination reactions. To quickly load MultiMate LR reaction prototype in APE, the following code can be copied and used in Tools/Recombination Reaction Editon/New reaction from clipboard:
MultiMate LR Reaction for APE:
{ApE recombination reaction:} {MultiMate LR Reaction (1-3-4-5-2)} {{{pMMK ENTR 1} {pMMK ENTR 2} {pMMK ENTR 3} {pMMK ENTR 4} DEST} {attB1 0 attB3 0 attB4 0 attB5 0 attB2 1}}
When imported as GenBank or Fasta files, the MultiMate LR reaction in APE will automatically recognize pMMK ENTR1-4 vectors and one DEST donors when launched through Tools/Recombination tools. For manual prediction of MultiMate assembly products, a list of the attL/R sequences and their attB products is provided in Supplementary Table 3.
Cre-mediated recombination of DNA elements
One acceptor and one or multiple donor vectors were assembled using Cre-mediated recombination as previously described26,32,47. One acceptor and one or more donors were mixed with a ratio of 1:1.1 in distilled H2O with 0.5 ul (7.5 U) of CRE recombinase (NEB # M0298M) and 1 ul Cre buffer (provided with CRE recombinase) to a final volume of 10 ul in distilled H2O. 500-1000 ng of total DNA were used for each reaction. Cre-reactions were incubated for 1 hour at 37°C, followed by heat inactivation at 70°C for 10 minutes. 2-3 ul were transformed into homemade electrocompetent Top10 E.Coli, followed by 2 hours recovery at 37°C and plating on LB/agar plates with the appropriate antibiotics. Cre-recombination products were predicted using Cre-ACEMBLER Vers. 2.0 47.
Cell culture methods
Human cells (HEK293T, HeLa, H4, RPE-1 hTERT and SH-SY5Y) were purchased from ATCC and propagated as adherent cultures in 60 or 100 cm dishes in a humidified incubator (37°C, 5% CO2). For passaging cells were washed with phosphate saline buffer (DPBS, Gibco # 14190144), detached using 0.25% Trypsin (Thermo Fisher #25200056) followed by a brief incubation at 37°C, centrifuged at 300x RCF and resuspended in fresh media in a new plate at the desired concentration. Suspension cultures of Sf21 insect cells were grown in 125 ml or 250 ml polycarbonate Erlenmeyer flasks with vent cap (CORNING, #431143, #431144) at 27°C in a shaking incubator. Sf21 were split every 2/3 days and maintained at concentrations between 0.5-2×106 cells/ml. Origin and media formulation recipe for each cell line is reported in Supplementary Table 4.
For transfection in HEK293T, 2 ⨯105 cells/well were seeded in multi-24 wells. Transfections were carried out using Polyfect (QIAGEN #301105), following manufacturer’s instructions. Briefly 500 ng of DNA were resuspended in 25 ul of Optimem (Gibco #31985062), supplemented with 5 ul Polyfect and incubated for 15 minutes at room temperature. Transfection mix was resuspended with 100 ul of complete media and added dropwise to each well. Cells were cultured for at least 48 hours before assessing the phenotype (e.g. fluorescence markers expression).
Baculovirus vector amplification
Assembled MultiMate vectors were shuttled on baculovirus genomes (bacmids) propagated in E.Coli using Tn7 transposition. 200-1000 ng of MultiMate vector were transformed in chemically competent DH10-MultiBacMam-VSV-G19, DH10-EMBacY32 or commercial DH10Bac (ThermoFisher # 10361012) as previously described32. Bacteria were streaked on LB/Agar plates with Gentamycin, Kanamycin, Tetracyclin, IPTG and Bluo-Gal and incubated for 24-hours for blue-white screening. White colonies were picked and grown overnight in 3 ml of LB supplemented with Gentamycin/Kanamycin to extract bacmid DNA through alkaline lysis/ethanol precipitation as previously described32,44.
For transfection in insect cells, 0.8-1×106 Sf21 cells/well were seeded on multi-6 well plates in 3 ml of Sf-900 II media. 10 ul of purified bacmid were resuspended in 130 ul Sf-900 II media with 10 ul X-treme XP transfection reagent (Roche # 06366236001) and incubated at room temperature for 15 minutes. The entire transfection mix was added dropwise to a single well and cells were incubated at 27°C in a static incubator. V0 viral stocks were harvested collecting the supernatant of transfected cells 72-96 hours post transfection as previously described19,32,47. 1-3 ml of V0 viral stocks was added to 10 ml of fresh Sf21 cells at 0.8×106 cells/ml. Cells were cultured in 50 ml Falcon tubes while shaking at 27°C and counted every day to monitor cell proliferation and size using Luna cell-counter (LogosBio). Successfully infected cells displayed arrested proliferation and increased average cell size (13-14 µm control, 16-20 µm infected). V1 viral harvest were collected as previously described 2 days after proliferation arrest (DPA+24)19,32,47 by centrifugation at 4500 x rcf. 500 µl/1 ml of V1 viral stocks was added to 50 ml of fresh Sf21 cells at 0.8×106 cells/ml in 125 ml Erlenmeyer flasks and cells were cultured at 27°C in a shaking incubator. V2 viral harvests were collected by centrifugation at 4500 x rcf, and concentrated 20 times by high-speed centrifugation at 11000 x rcf, followed by resuspension in DPBS supplemented with 3% heat inactivated FBS and 1 %Glycerol for storage at -80°C.
Baculovirus vector titration and transduction
For BV expressing fluorescent markers in human cells, titration was performed as previously described48. HEK293T were used to determine viral titers. Briefly 5⨯105 cells/well were seeded in multi-48 wells plates in 200 ul of complete media. Concentrated virus was serially diluted in DPBS and 50 µl were dispensed to each well.
Spinoculation (30’ at 600 x rcf at 27°C) was used to enhance transduction as previously reported48. 24 hours after transduction, cells were analysed using flow-cytometry to determine the percentage of transduced cells. TU/ml values from dilutions giving less than 20% transduction efficiencies were averaged to estimate the titer as previously described49 and using Supplementary Equation 1. For experiments in which different viral titers were used, multiplicity of transduction (MOT) was calculated as TU/Cn.
Confocal and widefield imaging
Confocal images were acquired using a Leica Sp8 equipped with 405, 458, 476, 488, 496, 514, 561, 594, 633 nm laser lines and 37°C stage. For time lapse confocal experiments on living cells the stage was supplemented with 5% CO2. For higher magnification, cells were plated on Lab-Tek borosilicate multi-8 wells (Thermo Fisher # 155411). For experiments in which transduced cells expressed more than 3 different fluorochromes, laser intensity and detection filters were adjusted to reduce spectral overlap using individual fluorescence controls transfections. Widefield and phase contrast images were acquired using a Leica DMI6000 equipped with excitation/emission filters optimised for DAPI, GFP, Rhodamine, Texas Red and Far red.
Flow cytometry analysis
For flow-cytometry analysis cells were trypsinised and resuspended in complete media supplemented with 3µM DRAQ7 (Abcam #ab109202) to counterstain dead cells. Cells were analysed on a BD Fortessa, fluorochromes were detected as follow: eGFP and EYFP (FITC-A), mCherry (PECF594-A), mTagBFP (BV421-A), DRAQ7 (AlexaFluor700-A). SSC-A and FSC-A were used to discriminate single cells and cell populations by size. FlowJo X was used to analyse FCS files. All data represented are percentages of live single cells (DRAQ7-).
PCR genotyping, Sanger sequencing and deconvolution
Genomic DNA was extracted with QIAamp DNA Mini Kit (QIAGEN # 51306) following manufacturer’s instruction. A list of the predicted gene editing outcome sequences and genotyping oligos is provided in Supplementary Table 2. PCRs were performed using KAPA2G Fast Genotyping mix (SigmaAldrich # KK5621) following manufacturer’s instruction. Amplicons were run on 0.8% agarose gels, purified using QIAquick Gel Extraction Kit (QIAGEN # 28706) and eluted in distilled ddH2O. For Sanger sequencing 15 ul of purified PCR at 5-10 ng/µl were mixed with 2 µl of diluted (10 µM) sequencing primer and sent to an external sequencing service (Eurofins). Electropherograms (.ab1) from parental and transduced cells were fed into ICE34 from Synthego for sequence deconvolution and indels/knock-in estimation.
Western blot analysis
Total protein extracts from HEK293T were obtained by lysing the cells with ice-cold RIPA Buffer (Thermo Fisher # 89901) supplemented with protease inhibitors (Thermo Fisher # 78429) for 30’ on ice. Insoluble material was pelleted by centrifugation at 16000 x rcf at 4°C for 5 minutes. Total protein extracts from insect cells were obtained as previously described32. Proteins concentration were determined using Nanodrop. 10 µg protein/sample were stained with Laemmli buffer, boiled at 95°C for 5 minutes, separated using pre-cast NuPage 4-12% Bis-Tris SDS-Gels (Thermo Fisher # NP0321BOX) and transferred to PVDF membranes using iBlot. Membranes were blocked with 5% non-fat dry milk in T-TBS (50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 0.5% Tween) for 1 hour at room temperature. Membranes were incubated with primary antibodies diluted 1:1000 in T-TBS 5% milk overnight at 4°C while rocking, followed by 2 T-TBS washes and incubation with HRP-conjugated secondary antibody diluted 1:2000 in T-TBS 5% milk for 1 hour at room temperature. Membranes were washed again with T-TBS and developed using Pierce ECL reagents (Thermo Fisher # 34579) following manufacturer’s instructions. Finally, membranes were imaged using MyECL Imager.
A list of the primary and secondary antibodies used is provided in Supplementary Table 5.
CCT/TriC complex purification
Recombinant MultiMate-CCT BV was produced as previously described32 and used to infect Sf21 insect cells at a cell density of 1.0×106/mL in Sf-900 II medium. Cells were harvested 72-96 h after proliferation arrest by centrifugation at 1,000 g for 15 minutes. Cell pellets were resuspended in lysis buffer (50 mM HEPES-NaOH, 200 mM KCl, 10 mM Imidazole, 20 % Glycerol, pH 7.5, supplemented with EDTA-free protease inhibitor (Sigma-Aldrich) and Benzonase (Sigma-Aldrich)) and lysed by short sonication. The lysate was cleared by centrifugation at 18,000 rpm, 4°C, in a F21-8×50y rotor (Thermo Fisher Scientific) for 60 minutes. The supernatant was loaded on a TALON column (Generon), equilibrated in TALON A buffer (50 mM HEPES-NaOH, 200 mM KCl, 10 mM Imidazole, 20 % Glycerol, pH 7.5) with a peristaltic pump. The column was washed with ten column volumes (CV) of TALON A buffer before eluting the bound protein complex with a step gradient of TALON B buffer (50 mM HEPES-NaOH, 200 mM KCl, 250 mM Imidazole, 20 % Glycerol, pH 7.5). The CCT protein complex was buffer exchanged in Heparin A buffer (50 mM HEPES-NaOH, 100 mM KCl, 10 % Glycerol, pH 7.5) while concentrating. It was then subjected to a Heparin column (GE Healthcare) and eluted with a 1M KCl gradient in Heparin A buffer. Fully formed complexes and disassembled subunits were separated on a Superose 6 10/300 column (GE Healthcare) equilibrated in SEC buffer (20 mM HEPES-NaOH, 200 mM KCl, pH 7.5, 1 mM DTT, 10% Glycerol). Peak fractions were pooled and concentrated and the purity of the CCT complex was analyzed by SDS-PAGE.
Electron microscopy
For electron microscopy, copper grids with carbon coating (300 mesh, Electron Microscopy Sciences) were glow discharged for 10 s, and 5 μL of purified CCT was placed on the grids for 1 min. Afterwards the grid was washed for 15 s and floated onto a drop of filtered 3 % uranyl acetate for 1 min. Excess solution on the grids was blotted off using filter paper between each step. Grids were visualized under a FEI Tecnai 20 transmission electron microscope (TEM), and digital micrographs were taken using a FEI Eagle 4Kx4K CCD camera. Particle picking and processing was performed using RELION 250,51, 2D class averages were generated without applying symmetry or reference models.
Data availability statement
All plasmid sequences are provided in Supplementary Table 1. MultiMate-CellCycle, MultiMate-Rainbow and MultiMate-HITI-2c ACTB reagents will be made available for distribution by Addgene. All other reagents are available from the authors upon reasonable request.
Author contributions
I.B. and F.A. conceived and designed the study with input from C.S. and M.S.D.. F.A. performed experiments and analysed results. F.A. and M.P. designed, implemented and validated DNA assembly protocols. C.T. performed protein purification and EM. J.C and P.M. produced and provided reagents. M.S.D., C.S. and I.B. supervised and guided the research. F.A. and I.B. wrote the manuscript with input from all authors.
Competing interest statement
I.B. declares shareholding in Geneva Biotech Sàrl and is inventor on patents and patent applications covering DNA methods and baculovirus reagents licensed to Geneva Biotech Sàrl.
Additional Information
Supplementary Information contains: Supplementary Methods detailing MultiMate DNA assembly and MultiMate-HITI-2c safe harbour assembly; Supplementary Tables providing sequences, gene editing outcomes and genotyping oligonucleotides sequences, cell lines and antibodies; Supplementary Equation and Supplementary Video.
Extended Data Figure Legends
Acknowledgement
We thank all members of the Berger, Schaffitzel and Dillingham teams and our academic and industrial collaborators for their contributions. We thank Daniel Fitzgerald (Geneva Biotech) for helpful discussions. We acknowledge generous support from GE Healthcare through a Discovery Research Grant (to I.B.), from BrisSynBio, a BBSRC/EPSRC Research Centre for synthetic biology at the University of Bristol (BB/L01386X/1), and from the Max Planck Bristol Centre for Minimal Biology. I.B. is Investigator of the European Research Council (ERC Advanced Grant DNA-DOCK, Project Nr. 834631).