Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus

Abstract

CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.

Figure 1.

MultiMate enables rapid modular assembly of multifunctional DNA circuitry for efficient baculovirus-vectored delivery in human cells. (A) MultiMate assembly platform in a schematic view. Symbols are listed (left panel). attL/R flanked DNA from ENTR plasmid modules (upper box, dashed) are assembled on MultiBac-DEST (lower box) and further combined by in vitro Cre-mediated recombination to generate multicomponent MultiMate plasmids, maximally eliminating prokaryotic backbone DNA sequences. MultiMate plasmids are integrated in BVs customized for efficient delivery in human cells (right panel). (B) Confocal live cell imaging of H4, HeLa, HEK293T and SH-SY5Y 48 hours after transduction with MultiMate-Rainbow BV (upper panel) evidencing in all cells homogeneous sustained expression and correct subcellular localization of H2B-iRFP (nucleus), GTS-mTagBFP (Golgi), mAG-β-catenin (membrane and adherens junctions), EYFP-Tubulin (microtubules), mTFP1-Actin (cytoskeleton), mito-mCherry (mitochondria) and CyOFP1-ER (endoplasmic reticulum). Scalebar, 20 μm. (C) Twelve-hours confocal time-lapse imaging of live HeLa cells transduced with MultiMate-CellCycle BV (snapshots, Supplementary Video 1). Transduced cells constitutively express H2B-iRFP for DNA imaging. mAG-hGeminin and mKO2-hCdt1 are stabilized in S/G2 and G1 cell cycle stages, respectively. Upper panel shows G1/S transition, lower panel shows a dividing cell (arrows indicate tracked cells). Scalebar, 10 μm. DNA elements, plasmid topology and cell cycle schematics are illustrated (upper panel).

Figure 2.

Baculovirus-vectored delivery of complete multicomponent CRISPR/Cas9 toolkits for homology independent targeted integration (HITI) in human cells. (A) ACTB C-terminal tagging strategy: homologous directed repair (HDR) and homology independent targeted integration (HITI-2c) elements within attL1/attR3 sites (triangles) and MultiMate-HITI-2c ACTB all-in-one DNA circuitry comprising Cas9, HITI-2c donor, sgRNA cassette, eGFP reporter. A module encoding AcrII4 Cas9 inhibitor under control of J23119 and polH promoters ensures vector stability. ACTB C-terminal exon is replaced with a synthetic exon, tagged with mCherry::T2A::puromycin. (B) Absolute gene editing efficiencies of HEK293T cells transfected or transduced with MultiMate-HDR or MultiMate-HITI-2c BVs in the absence of puromycin selection, at three- and ten-days post-transfection/transduction. (C) histogram of relative gene editing efficiencies normalized for transduction/transfection rates at 3 days post transfection/transduction (mCherry + cells %/eGFP + cells %). Histograms represent flow cytometry data. Mean ± s.d. of n = 3 independent biological replicates. ***P < 0.001, Student's t-test. (D–F) HEK293T 21 days after transduction with BV MultiMate HDR or MultiMate HITI-2c BVs after puromycin selection. (D) Representative flow-cytometry histograms. (E) Widefield microscopy, Scalebar = 20 μm. (F) Western blot of total protein extracts. Anti-β-actin antibody was used in top panel with anti-TUBULIN as loading control. (G) Confocal images of HEK293T, HeLa, H4 and SH-SY5Y cells 48 hours after transduction with MultiMate-HITI-2c BV. Scalebar is 50 μm. (H) Histograms of flow-cytometry data of HEK293T, HeLa, H4 and SH-SY5Y 72 hrs after transduction with MultiMate-HITI-2c VSV-G pseudotyped BVs, multiplicity of transduction (MOT) 1 and 10. Transduction efficiency = % of eGFP + cells; absolute gene editing efficiency = % of Cherry + cells. Mean ± s.d. of n = 3 independent biological replicates.

Figure 3.

Baculovirus-vectored safe-harbour homology-independent integration of large DNA cargoes in human genomes. (A) Safe-harbour HITI-2c strategy, HITI-2c payloads within attL1/attR3 sites (triangles) and MultiMate-HITI-2c ACTB all-in-one plasmid carrying Cas9, HITI-2c payload, sgRNA, AcrII4 and mTagBFP reporter. ACTB C-terminal exon is replaced with a synthetic exon, tagged with T2A::mCherry::P2A::puromycin (5′ integration marker), DNA insert ranging from 4.7 to 18 kb, and distinct 3′ integration markers (CMV Hygromycin (CH), CMV eGFP IRES Hygromycin (CGH) or EF1a EYFP-Tubulin IRES Hygromycin (EYH)). (B, C) HEK293T transduced with the indicated MultiMate-HITI-2c BVs after puromycin and hygromycin selection. (B) Confocal microscopy. Scalebar, 100 μm. (C) PCR genotyping. Oligonucleotide pairs (colour coded arrows) for each PCR are shown, their approximative position is depicted in (A). (D) Sanger sequencing of 5′ and 3′ genotyping PCRs (indicated by * in (C)) of HEK293T transduced with MultiMate-HITI-2c 18K-CGH BV. (E) Transduction efficiency (left histogram) and absolute gene editing efficiency (right histogram) of HEK293T and SH-SY5Y 72 hours after transduction with MultiMate-HITI-2c 18K-CGH VSV-G pseudotyped BV, derived from flow-cytometry data. Mean ± s.d. of n = 3 independent biological replicates. (F) Confocal microscopy pictures of HEK293T and SH-SY5Y 8 days after transduction with MultiMate-HITI-2c 18K-CGH VSV-G pseudotyped BVs in the absence of puromycin or hygromycin selection. (G) Representative flow-cytometry plots of HEK29T at 3- or 24-days post transduction with MultiMate-HITI-2c 18K-CGH VSV-G pseudotyped BV. (H) Representative flow-cytometry plots of HEK29T transduced with MultiMate-HITI-2c 18K-CGH VSV-G pseudotyped BV after puromycin (left) and puromycin/hygromycin selection (right).

Figure 4.

Safe-harbour large cargo integration of NPHS2 into patient-derived R138Q mutant podocin podocytes. (A) Safe harbour ACTB-NPHS2-Myc-Flag strategy. ACTB C-terminal exon is replaced with a P2A mCherry T2A Puro tagged synthetic exon (5′ integration marker), followed by CMV NPHS2-Myc-Flag and CMV Hygro (3′ integration marker). polH VSV-G and CMV mTagBFP are included in vector design to pseudotype and monitor viral transduction, respectively. (B–E) PM ciPods transduced with BV MultiMate HITI-2c NPHS2 in presence/absence of 24 h BacMam enhancer treatment analysed at 24 or 72 h post transduction. (B) transduction efficiencies (mTagBFP+ %); (C) mTagBFP mean fluorescence intensity levels, (D) absolute gene editing efficiencies (mCherry+ %). In (B–E) histograms represents means of flow-cytometry data, error bars are standard deviations of n = 3 independent replicates. (E) representative flow-cytometry histogram of mTagBFP intensity relative to (C). (F) Widefield microscopy images of PM ciPods at 24 h post-transduction in presence/absence of BacMam enhancer (top panel) or 36 days post-transduction following Puromycin/Hygromycin selection (bottom panel). (G) Immunofluorescence of unselected PM ciPods transduce with BV MultiMate HITI-2c NPHS2. Successfully edited (mCherry+) cells, display correct expression of NPHS2 through either α-Flag or α-NPHS2 antibodies. DAPI is used to counterstain nuclei. Scalebar = 20 μm. (H) Comparison of engineered PM ciPods with WT CiPods overexpressing NPHS2 R138Q under proliferative (33°C, top panel) or non-proliferative (10 days at 37°C, bottom panel) culturing conditions. α-Calnexin labels the endoplasmic reticulum, α-Myc is used to stain NPHS2. Scalebar = 20 μm.

Figure 5.

Highly efficient and prime-editing by using MultiMate all-in-one BV. (A) Schematic representation of MultiMate-PE2/PE3 HEK3 for trinucleotide (CTT) insertion. Plasmid DNA size (kb) is indicated. (B–D) HEK293T, RPE-1-hTERT, SH-SY5Y and HeLa transduced with MultiMate-PE2/PE3 HEK3 BVs. (B) transduction efficiencies (mTagBFP+ %) at 24 hours post-transduction, flow-cytometry data, error bars represent standard deviation of n = 3 independent replicates. (C) trinucleotide insertion efficiencies at 6 days post-transduction, sanger sequencing deconvolution data (ICE), error bars are standard deviations of n = 3 independent replicates. (D) representative Sanger sequencing alignment of data in (C). (E) Schematic representation of MultiMate-PE2/PE3 quadruplex trinucleotide insertion on DNMT1, EMX1, RNF2 and RUNX1. Plasmid DNA size (kb) is indicated, (F, G) HEK293T, RPE-1-hTERT, SH-SY5Y and HeLa transduced with MultiMate-PE2/PE3 quadruplex BVs, (E) transduction efficiencies (mTagBFP+ %) at 24 h post-transduction, flow-cytometry data, error bars represent standard deviation of n = 3 independent replicates. (F) trinucleotide insertion efficiencies at 6 days post-transduction, sanger sequencing deconvolution data (ICE), error bars are standard deviations of n = 3 independent replicates.

Figure 6.

HDR and HITI, but no prime editing, trigger excess backbone genomic integrations. (A–C) 29 days time-course monitoring of transfection and transduction marker in HEK293T transfected (A, B) or transduced (C,D) with the indicated all-in-one MultiMate constructs harbouring HDR/HITI (A, C) or PE2/3 quadruplex editing toolkits (B, D). A eGFP encoding plasmid or baculovirus, were used as controls for transfections (A, B) and transduction experiments (C, D) respectively. A dashed red line indicates control endpoint detection threshold. Mean with standard deviation of flow-cytometry data of n = 3 independent replicates. (E) Histogram of backbone associated fluorescence markers persistence at 29 days post-transduction normalised by the initial transfection or transduction efficiency. Mean with standard deviations of n = 3 independent replicates. ** P<0.01, ***P < 0.001, n.s.=not significant, Student's t-test. (F) Genomic DNA PCRs of HEK293T at 29 days post-transfection/transduction with the indicated constructs. ACTB amplicon was included to check for DNA quality. Gentamycin (Gent) amplicon detects integration events of both plasmids and baculoviruses, gp64 amplicon detects BV integration events only.