Main
Among current genome editing systems that function in both dividing and nondividing mammalian cells in vitro and in vivo, prime editing1 offers unusual versatility by enabling the replacement of a target DNA sequence with virtually any other specified sequence containing up to several hundred inserted, deleted or substituted base pairs2,3,4,5,6,7,8,9,10,11. This versatility makes PE systems particularly promising for the treatment of a broad range of genetic diseases in humans. A prime editor (PE) is an engineered protein consisting of a catalytically impaired programmable nickase domain (such as a Cas9 nickase) fused to an engineered reverse transcriptase (RT) domain. The prime editing guide RNA (pegRNA) specifies the target protospacer sequence and simultaneously encodes the desired edits in the reverse transcription template in the 3′ extension of the pegRNA. The mechanism of prime editing requires three independent nucleic acid hybridization events before editing can take place and does not rely on double-strand DNA breaks or donor DNA templates. As a result of this mechanism, prime editing is inherently resistant to off-target editing or bystander editing, and can proceed with few indel byproducts or other undesired consequences of double-strand DNA breaks1,12,13,14,15,16,17,18,19,20,21.
Fully realizing the potential of prime editing for research or therapeutic applications in mammals requires safe and efficient methods capable of delivering PEs into tissues in vivo. So far, several groups have reported the in vivo delivery of PE via viral delivery methods, including adenoviruses8 and adeno-associated viruses (AAV)8,9,10,11,12,22,23,24,25. Viral delivery methods, however, require that the transgene be encoded directly in the viral gene expression cassette, limiting transgene size. The AAV genome has a cargo gene size limitation of ~4.7 kb (not including inverted terminal repeats)26, requiring large cargoes such as PEs (6.4 kb in gene size for a first-generation PE) to be split into multiple AAVs25, limiting editing efficiency especially at moderate or low vector doses27. Viral delivery methods also pose potential safety risks including increased off-target editing from sustained transgene expression28 and the possibility of unwanted cargo DNA integration into host cell genomes29. Nonviral delivery methods, such as lipid nanoparticles, avoid some of these issues by packaging editors as transiently expressing messenger RNAs (mRNAs). In vivo nonviral targeting of tissues beyond the liver for efficient therapeutic gene editing remains a challenge30,31, however, despite recent advances targeting hematopoietic stem cells32.
Virus-like particles (VLPs) are potentially promising delivery vehicles that in principle offer key benefits of both viral and nonviral delivery methods33. VLPs are formed by spontaneous assembly and budding of retroviral polyproteins that encapsulate cargo molecules from producer cells. VLPs lack a packaged genome but retain the ability to transduce mammalian cells and release cargo34,35. Previous studies explored VLPs for delivering Cas9 nuclease36,37,38,39,40,41,42,43. We recently reported efficient in vivo delivery of adenine base editor (ABE):single guide RNA (sgRNA) ribonucleoproteins (RNPs) with iteratively engineered virus-like particles (eVLPs)44 that overcame specific molecular bottlenecks in cargo packaging, release and localization.
Engineered VLPs offer several advantages over other delivery methods as a candidate for in vivo PE delivery. First, eVLPs are not subject to stringent cargo size limitations, obviating the requirement of splitting PEs into multiple separate vectors. In addition, eVLPs can package RNPs, the most transient form of gene editing agents, thereby reducing frequency of off-target editing by minimizing the exposure duration of the genome to editing agents44,45,46. Since eVLPs lack DNA34,44, they avoid unwanted integration of viral genetic material into the genomes of transduced cells. Finally, eVLPs can be pseudotyped with different glycoproteins, enabling specific targeting of cell types of interest42 with envelope protein engineering efforts.
In this Article, we report the development of a PE-eVLP system that delivers complete PE systems including pegRNAs and nicking sgRNAs (ngRNAs) as RNPs. Simple replacement of base editors (BEs) with PEs in the optimized BE-eVLP system yielded very low functional delivery of prime editing systems ( T; p.R44X) (Fig. 5h), leading to diminishment of electroretinogram (ERG) responses from 3 weeks of age77. The corresponding mutation in humans in homozygous form causes Leber congenital amaurosis78. We previously demonstrated partial rescue of this disease phenotype using BE-eVLPs44. However, base editing at this site generates bystander edits at nearby A•T base pairs that may inactivate the RPE65 enzyme and cause adverse effects75, whereas the mechanism of prime editing inherently avoids bystander editing1.
We developed a PE3b strategy to correct the Rpe65 R44X mutation (Fig. 5h) by screening a panel of epegRNAs with varying primer binding site lengths (8–11 nt) and reverse transcription template lengths (11–25 nt), and seven candidate ngRNAs via plasmid transfection in an engineered NIH 3T3 cell line75 containing the corresponding mutation. The most promising PE3b strategy yielded 46% precise correction with 0.58% indels (Supplementary Table 5). We performed subretinal injection of 5-week-old rd12 mice with mutation-correcting v3 PE3b-eVLPs. HTS on the genomic DNA extracted from RPE tissues of the treated mice resulted in on average 7.2% correction of the mutation in bulk RPE tissue (Fig. 5i). This editing efficiency is higher than the reported value from triple AAV23 and low-dose dual-vector AAV-mediated PE delivery22 and comparable to that achieved by high-dose dual-vector AAV-mediated PE delivery22, but using a virus-free, single-particle delivery method.
We further analyzed Rpe65 transcripts in the extracted RNA by sequencing the complementary DNA. Editing was enriched at the transcript level, achieving on average 14% correction of the target mutation (Fig. 5j), presumably due to nonsense-mediated decay accelerating the degradation of uncorrected transcripts and enrichment of the major RPE65–expressing RPE cells that are preferentially transduced by PE-eVLPs among other cell types co-collected from tissue dissection. We confirmed robust expression of full-length RPE65 in v3 PE3b-eVLP-treated eyes by western blot (Fig. 5k). ERG of the v3 PE3b-eVLP-treated animals indeed showed substantial rescue of visual function in response to the stimuli compared to the untreated eyes (Fig. 5l,m). No off-target prime editing was detected above the 0.1% limit of detection at top ten CIRCLE-seq-nominated off-target sites associated with the rd12 epegRNA and ngRNA (Extended Data Fig. 10a,b). Together, these results demonstrate that in vivo application of optimized PE-eVLPs can correct a pathogenic mutation and partially rescue disease phenotype in mammals.
Discussion
Through extensive engineering of each major component, we developed an all-in-one virus-like particle that delivers PE RNPs into mammalian cells in culture and in vivo. Recent improvements to prime editing systems, including epegRNAs4, the PEmax architecture2 and MMR evasion2, contributed to improved outcomes with PE-eVLPs. Identification of bottlenecks in cargo packaging yielded PE variants that promote delivery by PE-eVLPs, as well as optimized eVLP architectures that facilitate cargo release and cargo localization. Introducing an additional mechanism for guide RNA recruitment addressed guide RNA packaging limitations, and an alternative v3b PE-eVLP system eliminated the need for covalent fusion to the Gag polyprotein. Together, these improvements yielded 170-fold higher average prime editing efficiency compared to v1.1 PE-eVLPs at a benchmark HEK3 test edit in HEK293T cells.
The optimized v3 and v3b PE-eVLPs systems proved efficacious in vivo. Potent prime editing was achieved in the mouse CNS via neonatal ICV injection, marking the first demonstration of CNS editing with transient delivery of a PE RNP. In the mouse retina, a single injection of v3 PE3-eVLPs precisely corrected a pathogenic 4-bp deletion in the rd6 model of retinal degeneration, restoring production of full-length MFRP protein. In the rd12 mouse model of genetic blindness, v3 PE3-eVLPs achieved comparable prime editing levels to a recently reported triple-vector AAV–PE system23, but using a nonviral, single-particle delivery vehicle, resulting in partial rescue of visual function. These findings demonstrate that v3 and v3b PE-eVLPs can achieve prime editing efficiency comparable to that attained using an AAV–PE delivery system, while avoiding drawbacks of viral delivery systems such as prolonged editor expression that increases off-target editing frequencies and the risk of oncogenic DNA integration29,79. To our knowledge, these findings also represent the first use of PE RNPs to achieve phenotypic rescue of an animal model of genetic disease.
While v3 and v3b PE-eVLPs demonstrated therapeutically relevant editing levels, PE-eVLP systems would benefit from the continued engineering effort for the next-generation PEs and improved eVLP systems. Furthermore, tissue-specific envelope protein engineering could expand the scope of PE-eVLP applications to diverse tissues. The possibility that single-dose, transient delivery of PE RNPs by PE-eVLPs may mitigate clinically relevant immunogenicity80 warrants further investigation. Lastly, future optimization in large-scale eVLP production will be necessary to fully realize the therapeutic potential of eVLPs. Nonetheless, the PE-eVLP system reported here offers unique advantages of nonviral, single-particle delivery of PEs in their most transient form as RNPs, presenting safety and target specificity advantages over DNA or mRNA delivery methods.
Methods
Molecular cloning
All plasmids were cloned using either USER, Gibson or Golden Gate assembly. DNA was PCR amplified with PhusionU Green Multiplex PCR Master Mix (Thermo Fisher Scientific, F564S). Plasmids were transformed into Mach1 (Thermo Fisher Scientific, C862003) chemically competent Escherichiacoli and were prepared using Plasmid Plus Midiprep kits (Qiagen, 12945).
Cell culture
HEK293T cells (ATCC, CRL-3216), Neuro-2a cells (ATCC, CCL-131) and Gesicle Producer 293T cells (Takara, 632617) were cultured in Dulbecco’s modified Eagle medium (DMEM) plus GlutaMax (Life Technologies; 10569044) supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were maintained at 37 °C with 5% CO2. Cell lines were confirmed to be negative for mycoplasma during this study.
PE-eVLP production
eVLPs were produced as previously described44. Briefly, Gesicle Producer 293T cells were plated at a density of 5 × 106 cells per flask in 10 ml of DMEM + 10% FBS media in T75 flask (Corning, 353136). A total of 18–24 h after seeding, a mixture of plasmids was transfected to producer cells with jetPRIME transfection reagent (Polyplus, 101000001) following the manufacturer’s protocol. For production of v3 PE-eVLPs, plasmids expressing VSV-G (400 ng), wild-type MMLV Gag–Pol (2,813 ng), Gag–MCP–Pol (1,125 ng), Gag–PE (563 ng) and MS2-guide RNA (4,400 ng MS2-epegRNA for v3 PE2-eVLP, 3520 ng MS2-epegRNA and 880 ng MS2-ngRNA for v3 PE3-eVLP) were co-transfected to each T75 flask. For production of v3b PE-eVLPs, plasmids expressing VSV-G (400 ng), wild-type MMLV Gag–Pol (2,813 ng), Gag–COM–Pol (2,000 ng), Gag–P3–Pol (422 ng), P4–PE (422 ng) and COM-gRNAs (4,400 ng COM-epegRNA for v3b PE2-eVLP, 3,520 ng COM-epegRNA and 880 ng COM-ngRNA for v3b PE3-eVLP) were co-transfected to each T75 flask. A total of 40–48 h after transfection, supernatants were collected, centrifuged at 500g for 5 min, then the supernatant was filtered through 0.45-μm polyvinylidene difluoride (PVDF) filter. For PE-eVLPs used with cultured cells, 5× PEG-it Virus Precipitation Solution (System Biosciences, LV825A-1) was subsequently added to the supernatant to precipitate eVLPs overnight at 4 °C. The next day, the eVLPs were pelleted by centrifugation at 1,500g for 30 min at 4 °C and were concentrated 100-fold by resuspending in 100 μl of Opti-MEM (Life Technologies; 31985070). All eVLPs tested for optimization experiments in cell culture were concentrated uniformly using the above mentioned method to facilitate direct comparison of PE-eVLP potency at the same volume of eVLPs transduced. PE-eVLPs concentrated by this method contain approximately 2.5 × 108 eVLPs μl−1. For PE-eVLPs used in vivo, eVLPs were concentrated using a 20% (w/v) sucrose in phosphate-buffered saline (PBS) cushion solution via ultracentrifugation at 26,000 rpm (141,000g for an rAV of 118.2 mm) for 2 h at 4 °C using an SW28 rotor in an Optima XPN Ultracentrifuge (Beckman Coulter). The eVLP pellets were resuspended in cold PBS solution following ultracentrifugation. The eVLP solution was further centrifuged at 1,000g for 5 min on a fixed-angle tabletop centrifuge to remove debris. eVLPs purified by ultracentrifugation and used for in vivo applications were resuspended in a minimum volume of PBS solution to maximize the dose of PE-eVLPs within the permitted volume of injection. For short-term storage, eVLPs were stored at 4 °C for up to 1 week. For long-term storage, eVLPs were stored at −80 °C and thawed on ice immediately before use. Repeated freeze–thaw was avoided.
PE-eVLP transduction in cultured cells and genomic DNA collection
Target cells were plated at a density of 30,000–35,000 cells per well in 48-well plates (Corning, 354509). A total of 18–24 h after seeding, PE-eVLPs were added to the media of target cells. Unless otherwise noted, cellular genomic DNA was collected 72 h after transduction as previously described44. Briefly, medium was removed from each well and cells were washed with 1× PBS. Then 130 μl of lysis buffer (10 mM Tris–HCl pH 8.0, 0.05% SDS and 25 μg ml−1 proteinase K) was added to each well. Following incubation at 37 °C for 1 h, the lysate was heated to 80 °C for 30 min and was used directly as an input for downstream HTS preparation.
HTS of genomic DNA samples
HTS was performed as described previously1. Primers used for the amplification of genomic loci and corresponding amplicons are listed in Supplementary Table 2. Briefly, 1–5 μl of cell lysate containing genomic DNA described above was used directly for the amplification of the target locus in the first round of PCR (PCR1). For base substitution edits, the target locus was amplified using Phusion U Green Multiplex PCR Master Mix (Thermo Fisher Scientific, F564S) under the following conditions: 98 °C (3 min); 30 cycles of 98 °C (10 s), 61 °C (20 s) and 72 °C (40 s); and 72 °C (2 min). For insertion and deletion edits that are more susceptible to PCR bias, PCR1 was monitored using SYBR Green fluorescence with qPCR and the reaction was stopped at the exponential phase to avoid over-amplification of the target locus. Subsequently, 1–2 μl of PCR1 product was used as a template for the second round of PCR (PCR2) to append unique Illumina barcodes. PCR2 was conducted using Phusion U Green Multiplex PCR Master Mix under the following condition: 98 °C (3 min); 10 cycles of 98 °C (10 s), 61 °C (20 s) and 72 °C (30 s); and 72 °C (2 min). PCR2 products were pooled and purified on 1.5 % agarose gel by gel extraction using QIAquick Gel Extraction Kit (Qiagen; 28704). The library was quantified by Qubit dsDNA HS Assay Kit (Thermo Scientific, Q32852) and was sequenced using Illumina MiSeq 300 v2 Kit (Illumina) on Illumina MiSeq instrument.
HTS data analysis
HTS reads were demultiplexed using the MiSeq Reporter software v2.6 (Illumina). Data analysis was conducted using CRISPResso2 as previously described2. Briefly, reads were filtered by minimum average quality score (Q > 30) before analysis. CRISPResso2 analysis was performed with ‘discard_indel_reads’ on, and the quantification window was set to encompass at least ten nucleotides upstream and downstream of the pegRNA and/or ngRNA nick site. Prime editing efficiency was calculated as the percentage of reads with the desired editing without indels divided by the total number of reference-aligned reads. Indel frequency was calculated as the number of discarded reads divided by the total number of reference-aligned reads. The lower limit of detection is assumed to be 0.1%, defined by the error rate of the HTS method used.
Plasmid transfection
Plasmid transfection for purposes other than eVLP production was performed using Lipofectamine 2000 (Invitrogen, 11668500) following the manufacturer’s protocol as described previously1,2. Briefly, cells were seeded in either 96-well plates (Corning, 353075) at a density of 15,000–20,000 cells per well or 48-well plates (Corning, 354509) at a density of 30,000–35,000 cells per well. A total of 16–24 h after seeding, test plasmids were mixed in Opti-MEM (Life Technologies, 31985070). For 96-well transfection, editor plasmids (250 ng) and guide RNA plasmids (40 ng epegRNA for PE2; 30 ng pegRNA and 10 ng ngRNA for PE3) were mixed with 0.5 μl of Lipofectamine 2000. For 48-well transfection, editor plasmids (750 ng) and guide RNA plasmids (250 ng epegRNA for PE2; 188 ng epegRNA and 62.5 ng ngRNA for PE3) were mixed with 1 μl of Lipofectamine 2000. Following incubation at room temperature for 10 min, the transfection mixture was added directly to the media of the target cells. Genomic DNA was collected 72 h after transfection following the protocol described above.
PE-eVLP protein content quantification by ELISA
The protein content of PE-eVLPs was quantified as described previously44. Briefly, PE-eVLPs used for protein content quantification were concentrated via ultracentrifugation as described above for optimal detection of protein. A total of 5 μl of ultracentrifuged PE-eVLPs was mixed with 2× dye-free Laemmli sample buffer (100 mM Tris pH 7.5, 4% SDS and 20% (v/v) glycerol) and was incubated at 95 °C for 15 min. The lysed PE-eVLPs were used as input for quantification of PE protein and MLV p30 protein by ELISA. PE content in PE-eVLPs was quantified using the FastScan Cas9 (Streptococcuspyogenes) ELISA kit (Cell Signaling Technology, 29666C) following the manufacturer’s protocol. A standard curve was generated using recombinant Cas9 (S. pyogenes) nuclease protein (New England Biolabs, M0386). The number of eVLPs per volume was measured by quantifying MLV p30 content with the MuLV Core Antigen ELISA kit (Cell Biolabs, VPK-156) following the manufacturer’s protocol and calculated by assuming that 20% of the measured p30 in solution is associated with VLPs and that each VLP molecule contains 1,800 molecules of p30 (ref. 81).
PE-eVLP pegRNA content quantification by RT–qPCR
PE-eVLPs used for pegRNA content quantification were concentrated via ultracentrifugation as described above. A total of 10 μl of ultracentrifuged PE-eVLPs was treated with DNase I (Qiagen, 79254) to remove any residual plasmid DNA carry-over. RNA was extracted from PE-eVLPs using QIAamp Viral RNA Mini Kit (Qiagen, 52906) following the manufacturer’s protocol.
For standard curve generation, guide RNAs (epegRNAs or ngRNAs) were transcribed in vitro using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, E2040S) following the manufacturer’s protocol. RNA was purified using Monarch RNA Cleanup Kit (New England Biolabs; T2030S). In vitro-transcribed epegRNAs were subjected to the same DNase treatment and RNA extraction procedure as PE-eVLPs as described above.
Standard and test gRNAs extracted from PE-eVLPs were serially diluted and reverse-transcribed to generate cDNA using SuperScript IV Reverse Transcriptase (Invitrogen, 18090010) following manufacturer’s protocol. Briefly, a sequence-specific reverse primer that binds the 3′ end of gRNAs was annealed to the template RNA upon incubation at 65 °C for 5 min. RT mix was then added to the annealed RNA, and the reaction was incubated at 65 °C for 20 min, followed by 80 °C for 10 min. The cDNA generated was used as an input for qPCR. qPCR was performed using Power SYBR Green Master Mix (Applied Biosystems, 4368577) under the following condition: 95 °C (10 min), and 40 cycles of 95 °C (15 s) and 67 °C (1 min). Because all RNAs including gRNAs are potentially susceptible to degradation in cells, to exclusively quantify functional epegRNAs that retain their spacer, scaffold and 3′ extension, qPCR primers were designed to anneal to part of the spacer and scaffold at the 5′ end, and to part of the PBS and structured motif at the 3′ end. RT–qPCR primers are listed in Supplementary Table 3.
DLS
DLS was performed with a Zetasizer Nano ZS (Malvern Panalytical). A total of 5 μl of PE-eVLPs purified by ultracentrifugation were diluted in 800 μl of PBS, and the samples were transferred to cuvettes for measurement. Backscatter (173°) measurements (n = 3 per sample) were taken each using ten runs of 8 s and an equilibration time of 10 s. The number size distribution was calculated using an estimated refractive index of 1.45 and absorption of 0.001 based on the preset values for proteins and phospholipids, and mean diameter reported represents the average size of three technical replicates.
Western blot analysis of producer cell lysate protein content
Gesicle Producer 293T cells were plated at a density of 300,000–320,000 cells per well in six-well plates (Corning; 3506). A total of 16–24 h later, wild-type Gag–Pol plasmids (6,000 ng) and editor fusion plasmids (2,000 ng) were mixed with 8 μl of Lipofectamine 2000. A total of 48 h later, cells were washed with PBS and lysed in 200 µl of RIPA buffer (supplied by Broad Institute Internal Store; 2.5 mM sodium deoxycholic acid, 1 mM EDTA, 1% Triton X-100, 500 mM NaCl, 20 mM Tris–HCl pH 8 and 0.1% SDS in RODI water) supplemented with 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, 93482) and cOmplete Protease Inhibitor (Sigma-Aldrich, 4693159001) by incubating at 4 °C for 30 min. The lysate was centrifuged at 12,000 rpm (13,700g) for 20 min and the supernatant was collected. Total protein level was measured by bicinchoninic acid assay (Thermo Scientific, #23252) following the manufacturer’s protocol and samples were normalized on the basis of the protein concentration measured.
Western blots were performed as described previously44. Briefly, lysates were separated on a NuPAGE 3–8% Tris-acetate gel (Thermo Fisher Scientific; EA0376) in NuPAGE Tris-acetate SDS running buffer (Thermo Fisher Scientific, LA0041) for 45 min at 150 V. The gel was transferred to a PVDF membrane (Life Technologies, IB24002) using an iBlot2 Gel Transfer Device (Thermo Fisher Scientific, IB21001) at 20 V for 7 min. The membrane was blocked using Intercept Blocking Buffer (LI-COR, 927-70050) for 1 h at room temperature with gentle rocking. The membrane was washed three times with 1x TBS-Tween by rocking at room temperature for 5 min per wash. Then the membrane was incubated with primary antibodies (mouse Cas9 antibody: Thermo Fisher Scientific, #MA5-23519; rabbit GAPDH antibody: Cell Signaling Technology, #2118) at 1:1,000 dilution in Superblock Binding Buffer (1% bovine serum albumin (BSA) in TBS-Tween). The next day, the membrane was washed three times with 1× TBS-Tween as described above. Then the membrane was incubated with secondary antibodies (goat anti-mouse antibody: LI-COR IRDye 680RD and 926-68070, and goat anti-rabbit antibody: LI-COR IRDye 800RD and 926-32211) at 1:10,000 dilution in Superblock Binding Buffer for 1 h at room temperature with gentle rocking. The membrane was washed three times before imaging using a ChemiDoc MP Imaging System (Bio-Rad, 12003154).
Western blot analysis of PE-eVLP protein content
PE-eVLPs were concentrated via ultracentrifugation and lysed in 2× dye-free Laemmli buffer as described before. Western blots were performed as described above, using mouse Cas9 antibody (Thermo Fisher Scientific, MA5-23519) as the primary antibody and goat anti-mouse antibody (LI-COR IRDye 680RD and 926-68070) as the secondary antibody.
Off-target analysis in cultured cells
For comparison of off-target editing between plasmid transfection and PE-eVLPs, cells were seeded at a density of 30,000–35,000 cells per well in 48-well plates as described previously. After 1 day, plasmid transfection was performed as described previously and PE-eVLP transduction was performed by adding 10 μl of ultracentrifuge-concentrated PE-eVLPs to media containing target cells. A total of 3 days after treatment, cells were split into new 48-well plates to prevent cells from being overconfluent. A total of 7 days after treatment, genomic DNA was extracted from cells as described previously. Genomic DNAs were used for the amplification of the on-target HEK4 locus, off-target site 1 and off-target site 3.
The off-target editing was analyzed as described previously2. Briefly, reads were aligned to reference off-target amplicons using CRISPResso2 with parameters ‘-q 30’, ‘discard_indel_reads TRUE’ and ‘-w 25’. Off-target reads were called as leniently as possible to capture all potential reverse transcription products at the Cas9 nick site. To assess potential pegRNA-mediated off-target editing, nucleotide sequence 3′ of the Cas9 nick site was compared to the 3′ DNA flap sequence encoded by the epegRNA reverse transcription template. The minimum sequence of the 3′ DNA flap that deviates from Cas9 nick site was designated as an off-target marker sequence. All reference-aligned reads that contain this off-target marker sequence were called as off-target reads and pegRNA-mediated off-target editing efficiency was calculated as the percentage of (reads containing the off-target marker sequence)/(the total number of reference-aligned reads). Frequency of insertions or deletions at the off-target Cas9 nick sites were quantified as a percentage of (discarded reads)/(the total reference-aligned reads). Total off-target editing is calculated as (pegRNA-mediated off-target editing frequency) + (indel frequency at the Cas9 nick site).
Lentivirus production
Lentivirus used in this study was produced as described previously44. Briefly, HEK293T/17 (ATCC CRL-11268) cells were plated in T75 flasks (Corning; 353136) at a density of 5 × 106 cells per flask in 10 ml of DMEM + 10% FBS medium. A total of 20–24 h after seeding, for production of lentivirus expressing GFP:KASH, plasmids expressing VSV-G (6,000 ng), psPAX2 (9,000 ng) and lenti-GFP:KASH (9,000 ng) were mixed in 1.5 ml of Opti-MEM and were incubated with FuGENE HD Transfection Reagent (Promega; E2312) following the manufacture’s protocol. The plasmid transfection mixture was added directly to the media of the cells. A total of 40–48 h after transfection, supernatants were collected and centrifuged at 500g for 5 min to remove the cell debris. Then the supernatant was filtered through 0.45-μm PVDF filter. Lentivirus was subsequently concentrated into 20% (w/v) sucrose in PBS cushion solution via ultracentrifugation as described above for eVLP production.
Animals
Timed pregnant C57BL/6J mice for P0 studies were purchased from Charles River Laboratories (027). Retinal degeneration mouse models rd6 (003684) and rd12 (005379) were purchased from the Jackson Laboratory. All experiments involving live animals were approved by the Broad Institute Institutional Animal Care and Use Committee (D16-00903; 0048-04-15-2) and the University of California, Irvine Institutional Animal Care and Use Committee (D16-00259; AUP-21-096). Mouse housing facilities were maintained at 20–22 °C with 30–50% humidity, on a 12 h light/12 h dark cycle with ad libitum access to standard rodent diet and water. Animals were randomly assigned to various experimental groups.
P0 ICV injections and tissue collection
P0 ICV injections were performed as previously described25,44,82. Briefly, syringes for microinjection were generated by pulling PCR Micropipettes (Drummond Scientific Company, 5-000-1001-X10) on the Sutter P1000 micropipette puller. Injection solution was made immediately before injection by mixing 4 μl of PE-eVLPs, 0.3 μl of VSV-G pseudotyped GFP:KASH lentivirus and 0.1 μl of Fast Green. A total of 4 μl injection solution (containing approximately 1.0 × 1011 eVLPs) was front-loaded to Drummond PCR pipettes. Neonatal mice were cryo-anesthetized on ice until they were unresponsive to bilateral toe pinch. Then 2 μl of injection solution was injected into each ventricle. Injection was verified by the spread of Fast Green via transillumination of the head. A total of 3 weeks after injection, mice were killed by CO2 asphyxiation. Brain tissues were collected by splitting the hemispheres along the sagittal plane.
Nuclear isolation and sorting
Nuclei isolation was performed as previously described25,44,82. Briefly, collected brain hemispheres were transferred to the Dounce homogenizer (Sigma-Aldrich, D8938) along with 2 ml of EZ-PREP buffer (Sigma-Aldrich, NUC-101). Tissues were homogenized with 20 strokes with pestle A and 20 strokes with pestle B. The homogenates were combined with 2 ml of fresh EZ-PREP buffer and were centrifuged at 500g for 5 min. Supernatant was decanted and the nuclei pellet was washed by resuspending in 4 ml of ice-cold Nuclei Suspension Buffer (100 μg ml−1 BSA and 3.33 μM Vybrant DyeCycle Ruby (Thermo Fisher, V10309) in PBS). The mixture was centrifuged again at 500g for 5 min. Following two rounds of wash total, the pellet was resuspended in 3 ml of nuclear resuspension buffer and was filtered through 35-μm cell strainer. The isolated nuclei were flow-sorted using the Sony MA900 Cell Sorter (Sony Biotechnology) at the Broad Institute flow cytometry core using MA900 Cell Sorter software v3.1. See Extended Data Fig. 7 for a representative example of fluorescence-activated cell sorting gating. Nuclei were sorted into DNAdvance lysis buffer (Beckman Coulter, A48705) supplemented with 25 mM dithiothreitol and Proteinase K (Thermo Fisher). The genomic DNA was subsequently purified following the manufacturer’s protocol using DNAdvance kit (Beckman Coulter, A48705). For neuron-specific sorting, nuclei isolation was performed as described above. After the first centrifugation step, nuclei were washed with 4 ml of PBS + BSA (100 μg ml−1). Following centrifugation and decanting supernatant, nuclei were resuspended with 1 ml of PBS + BSA (100 μg ml−1) and 1 μl of anti-NeuN antibody (Abcam, ab190565) was added. Following incubation at 4 °C for 45 min in the dark with rocking, the mixture was centrifuged at 500g for 5 min. The supernatant was decanted and the pellet was washed twice with 1 ml of PBS supplemented with 100 μg ml−1 BSA and 3 μM DAPI (Thermo Fisher, D1306). The stained nuclei were then flow-sorted and processed as described above.
Subretinal injection
The injection mix for subretinal injection was prepared immediately before injection by mixing 15–20 μl of PE-eVLP with 0.3 μl AAV–GFP (Addgene, 105530-AAV1). Mice were anesthetized by intraperitoneal injection of a cocktail consisting of 20 mg ml−1 ketamine and 1.75 mg ml−1 xylazine in PBS at a dose of 0.1 ml per 20 g body weight, and their pupils were dilated with topical administration of 1% tropicamide ophthalmic solution (Akorn, 17478-102-12) and 10% phenylephrine (Valeant, 42702-0103-05). Subretinal injections were performed under an ophthalmic surgical microscope (Zeiss). The corneas were hydrated with the application of GenTeal Severe Lubricant Eye Gel (0.3% hypromellose, Alcon). An incision was made through the cornea adjacent to the limbus at the nasal side using a 27-gauge needle. A 34-gauge blunt-end needle (World Precision Instruments, NF34BL-2) connected to an RPE-KIT (World Precision Instruments, RPE-KIT) by SilFlex tubing (World Precision Instruments, SILFLEX-2) was inserted through the corneal incision while avoiding the lens and advanced through the retina. Each mouse was injected with 1 μl of PE-eVLP (containing approximately 4.2 × 1010 eVLPs) + AAV1–GFP (used to confirm injection efficiency) mixture per eye. After injections, the gel was reapplied, anesthesia was reversed with intraperitoneal atipamezole (2.5 mg kg−1; MWI Animal Health, 032800) and mice were allowed to recover on a heat pad. Two weeks after injection, GFP signal was assessed by scanning laser ophthalmoscopy as a marker for injection efficiency and retinas that showed>80% GFP+ were collected for downstream analysis.
Electroretinography
Before recording, mice were dark adapted for 24 h overnight. Under a safety light, mice were anesthetized by intraperitoneal injection of a cocktail consisting of 20 mg ml−1 ketamine and 1.75 mg ml−1 xylazine in PBS at a dose of 0.1 ml per 20 g body weight, and their pupils were dilated with topical administration of 1% tropicamide ophthalmic solution (Akorn, 17478-102-12) and 10% phenylephrine (Valeant, 42702-0103-05). The corneas were hydrated with the application of GenTeal Severe Lubricant Eye Gel (0.3% hypromellose, Alcon). The mouse was placed on a heated Diagnosys Celeris rodent ERG device (Diagnosys LCC). Ocular electrodes were placed on the corneas, the reference electrode was positioned subdermally between the ears, and the ground electrode was placed in the rear leg. The eyes were stimulated with a green light (peak emission 544 nm, bandwidth ∼160 nm) stimulus of −0.3 log (candela second per meter squared (cd s m−2)). The responses for ten stimuli with an inter-stimulus interval of 10 s were averaged together, and the a- and b-wave amplitudes were acquired from the averaged ERG waveform. The ERGs were recorded with the Celeris rodent electrophysiology system (Diagnosys LLC) and analyzed with Espion V6 software (Diagnosys LLC).
RPE dissociation and genomic DNA and RNA preparation
Under a light microscope, mouse eyes were dissected to separate the posterior eyecup (containing RPE, choroid and sclera) from the retina and anterior segments. Each posterior eyecup was immediately immersed in PBS. RPE, choroid and scleral cells were detached in PBS from the posterior eyecup by gentle pipetting, followed by a removal of the remaining posterior eyecup. Cells from rd6 mice were then processed for genomic DNA using the DNeasy Blood & Tissue Kit (Qiagen, 69504) and cells from rd12 mice were processed with the AllPrep DNA/RNA Micro Kit (Qiagen, 80284).
Western blot analysis of mouse RPE tissue extracts
To prepare the protein lysate from the mouse RPE tissue, the dissected mouse eyecup, consisting of RPE, choroid and sclera, was transferred to a microcentrifuge tube containing 40 μl of RIPA buffer with protease inhibitors and homogenized with a motorized grinder (Fisher Scientific, K749540-0000), incubated on ice for 20 min and then centrifuged for 20 min at 21,000g at 4 °C. The resulting supernatant was precleared with Dynabeads Protein G (Thermo Fisher, 10003D) to remove immunoglobulin contaminants from the blood before gel loading. A total of 10 μl of RPE lysates premixed with NuPAGE LDS Sample Buffer (Thermo Fisher, NP0007) and NuPAGE Sample Reducing Agent (Thermo Fisher, NP0004), and denatured at 70 °C for 10 min, was loaded into each well of a NuPAGE 4–12% Bis-Tris gel (Thermo Fisher, NP0321BOX), separated for 1 h at 130 V and transferred onto a PVDF membrane (Millipore, IPVH00010). After 1 h blocking in 5% (w/v) nonfat milk in PBS containing 0.1% (v/v) Tween-20 (PBS-T), the membrane was incubated with primary antibody, goat anti-mouse MFRP monoclonal antibody (1:1,000; R&D Systems, AF3445) or mouse anti-mouse RPE65 (1:1,000; in-house production83) diluted in 1% (w/v) nonfat milk in PBS-T overnight at 4 °C. After overnight incubation, membranes were washed three times with PBS-T for 5 min each and then incubated with donkey anti-goat IgG–horseradish peroxidase (HRP) antibody (1:10,000; Abcam, ab97110) or goat anti-mouse IgG–HRP antibody (1:5,000; Cell Signaling Technology, 7076S) for 1 h at room temperature. After washing the membrane three times with PBS-T for 5 min each, protein bands were visualized after exposure to SuperSignal West Pico Plus Chemiluminescent substrate (Thermo Fisher; 34577). Membranes were stripped (Thermo Fisher, 21059), reblocked and reprobed for β-actin expression using rabbit anti-β-actin polyclonal antibody (1:1,000; Cell Signaling Technology, 4970S), following the same protocol. The corresponding secondary antibody was goat anti-rabbit IgG–HRP antibody (1:5,000; Cell Signaling Technology, 7074S).
Immunohistochemistry of RPE flatmounts and cryosections
Mouse eyes were enucleated and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and washed three times in PBS for 5 min each. To make RPE flatmounts, the anterior segment and retina were removed from the posterior eyecup under a dissecting microscope, and four radial cuts were made toward the optic nerve to flatten the eyecup into an RPE flatmount. Samples were permeabilized and blocked in 0.1% Triton X-100 (Sigma-Aldrich, T8532) with 3% normal donkey serum (NDS) in PBS for 30 min and incubated with the appropriate primary antibody in PBS, 0.1% Triton X-100 and 3% NDS, including goat anti-MFRP antibody (1:100; R&D Systems, AF3445) and rabbit anti-ZO-1 polyclonal antibody (1:100; Invitrogen, 61-7300) overnight at 4 °C. The next day, samples were washed three times in PBS for 5 min each and then incubated with the appropriate secondary antibody in PBS + 0.1% Triton X-100 and 3% NDS, including Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:200; Thermo Fisher, A21207) and Alexa Fluor 647-conjugated donkey anti-goat IgG (1:200; Thermo Fisher, A32849) for 2 h at room temperature in the dark. Cryosection samples were incubated in 1 ml DAPI (Thermo Fisher, 62248) in PBS for 10 min. Samples were washed three times in PBS for 5 min each. The samples were then mounted with VECTASHIELD HardSet Antifade Mounting Medium (Vector Labs H-1400-10) and imaged on a Keyence BZ-X800 All-in-One fluorescence microscope.
CIRCLE-seq nomination of off-target sites for the rd6 and rd12 models
CIRCLE-seq off-target editing analysis was performed as previously described76,84. Genomic DNA from rd6 mouse liver was isolated using Gentra Puregene Kit (Qiagen, 158845) following the manufacturer’s instructions. Purified genomic DNA was sheared with a Covaris S2 instrument to an average length of 300 bp. The fragmented DNA was end repaired, A-tailed and ligated to a uracil-containing stem–loop adaptor, using the KAPA HTP Library Preparation Kit, PCR Free (KAPA Biosystems, KK8235). Adaptor-ligated DNA was treated with Lambda Exonuclease (New England Biolabs, M0262) and E. coli Exonuclease I (New England Biolabs, M0293) and then with USER enzyme (New England Biolabs, M5505) and T4 poly-nucleotide kinase (New England Biolabs, M0201). Intramolecular circularization of the DNA was performed with T4 DNA ligase (New England Biolabs, M0202) and residual linear DNA was degraded by Plasmid-Safe ATP-dependent DNase (Lucigen, E3110). Synthetic guide RNAs were ordered from IDT with standard 2′-O-methyl modification at first three and last three bases. The synthetic guide RNAs were resuspended to 9 µM in nuclease-free water, denatured at 90 °C for 5 min and slowly annealed at 0.1 °C s−1 to 25 °C. In vitro cleavage reactions were performed with 125 ng Plasmid-Safe-treated circularized DNA, 90 nM Cas9 nuclease protein (New England Biolabs, M0386) and 270 nM synthetic guide RNA in a 50 µl volume for 1 h. Cleaved products were treated with proteinase K as described84, A-tailed, ligated with a hairpin adaptor (New England Biolabs, E7600S), treated with USER enzyme (New England Biolabs, M5505) and amplified by PCR with barcoded universal primers (New England Biolabs, E7600S) using Kapa HiFi Polymerase (KAPA Biosystems, KK4824). Libraries were sequenced with 150-bp/150-bp paired-end reads with an Illumina MiSeq instrument. CIRCLE-seq data analyses were performed using open-source CIRCLE-seq analysis software and default recommended parameters85. The top ten nominated off-target sites for epegRNA used for the rd6 and rd12 models were analyzed by HTS from the RPE tissue of untreated or v3 PE3b-eVLP-treated mice. Off-target editing for epegRNA-associated off-target sites was analyzed, as described above, as (pegRNA-mediated off-target editing frequency) + (indel frequency at the Cas9 nick site). Insertions or deletions at ngRNA-associated off-target sites were analyzed as a percentage of discarded reads divided by the total reference-aligned reads. Top ten CIRCLE-seq nominated off-target sites are listed in Supplementary Table 6 (rd6 model) and Supplementary Table 7 (rd12 model).
Statistics and reproducibility
Data are presented as mean and standard error of the mean (s.e.m.). Comparisons of different versions of PE-eVLPs were made with eVLPs produced and transduced in parallel in one large experiment. Biological replicates were obtained by treating three independently maintained cell line splits (aliquots) for cell culture studies, or three or more animals for in vivo studies, with a single batch of PE-eVLPs. Low batch-to-batch variability for different PE-eVLP batches is shown in Extended Data Fig. 5b. The sample size and the statistical tests used for each experiment are described in the figure legends. No statistical methods were used to predetermine sample size. Statistical analysis was performed using GraphPad Prism software.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
HTS data files were deposited to the NCBI Sequence Read Archive database under accession codes PRJNA980181 (ref. 86). DNA sequences of the PE-eVLP architecture are provided in Supplementary Information. The following key plasmids from this work are deposited to Addgene for distribution: Gag–MCP–Pol (Addgene #211370), Gag–PE (Addgene #211371), MS2-epegRNA–Dnmt1 (Addgene #211372), Gag–COM–Pol (Addgene #211373), Gag–PE3–Pol (#211374), P4–PE (#211375), COM-epegRNA–Dnmt1 (#211376). Other plasmids and raw data are available from the corresponding author on request. Unmodified image of the western blots shown in Fig. 5d,k are provided as Source data. Source data are provided with this paper.
Code availability
The code used for analysis of HTS data is available at https://github.com/pinellolab/CRISPResso2. The code used for analysis for CIRCLE-seq data is available at https://github.com/tsailabSJ/circleseq.
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Acknowledgements
This work was supported by NIH UG3AI150551, U01AI142756, R35GM118062 and RM1HG009490; the Bill and Melinda Gates Foundation; and HHMI. This research was supported in part by NIH research grants EY034501 (NEI) and a grant from Foundation Fighting Blindness (TRAP program) to K.P. and NEI research grants to F30EY033642 and T32GM008620 to S.W.D. The authors acknowledge support from a NIH core grant P30 EY034070, UCI School of Medicine Dean’s office grant and from a Research to Prevent Blindness unrestricted grant to the Department of Ophthalmology at UCI. A.R. was supported by an NSF Graduate Research Fellowship. We thank Michael Howard, Kenia Guzman and the Broad Vivarium staff for advice and assistance with mouse husbandry. We thank J. Doman, S. DeCarlo, P. Randolph, A. Yan and A. Tworak for helpful discussions. This article is subject to HHMI’s Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication.
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Authors and Affiliations
Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA
Meirui An, Aditya Raguram, Samagya Banskota, Jessie R. Davis, Gregory A. Newby, Paul Z. Chen & David R. Liu
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA
Meirui An, Aditya Raguram, Samagya Banskota, Jessie R. Davis, Gregory A. Newby, Paul Z. Chen & David R. Liu
Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA
Meirui An, Aditya Raguram, Samagya Banskota, Jessie R. Davis, Gregory A. Newby, Paul Z. Chen & David R. Liu
Gavin Herbert Eye Institute, Center for Translational Vision Research, Department of Ophthalmology, University of California, Irvine, CA, USA
Samuel W. Du & Krzysztof Palczewski
Department of Physiology and Biophysics, University of California, Irvine, CA, USA
Samuel W. Du & Krzysztof Palczewski
Department of Chemistry, University of California, Irvine, CA, USA
Krzysztof Palczewski
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA
Krzysztof Palczewski
Contributions
M.A. designed and executed in vitro and in vivo experiments described in this work, and analyzed data. A.R. and S.B. advised and designed initial experiments. S.W.D. performed subretinal injection of rd6 and rd12 mice and performed phenotypic assessment. M.A. and J.R.D. performed P0 ICV injection and tissue processing. G.A.N. and M.A. performed CIRCLE-seq. P.Z.C. assisted with DLS measurements. M.A. and D.R.L. designed the research and drafted this manuscript with input from all authors.
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Competing interests
The authors declare competing financial interests: M.A., A.R., S.B. and D.R.L. have filed patent applications on this work through the Broad Institute. S.B. is currently a consultant for Nvelop Therapeutics. J.R.D. is currently an employee of Prime Medicine. K.P. is a consultant for Polgenix, Alnylam and AbbVie, Inc. D.R.L. is a consultant and/or equity owner for Prime Medicine, Beam Therapeutics, Pairwise Plants, Chroma Medicine and Nvelop Therapeutics, companies that use or deliver genome editing or epigenome engineering agents. The remaining authors declare no competing interests. Correspondence: [email protected].
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Extended data
Extended Data Fig. 1 MMR-evading edits support more efficient prime editing.
Installation of nearby mutations improves prime editing efficiencies of v2.3 PE-eVLPs at the HEK3 locus and Dnmt1 locus in HEK293T and N2A cells respectively. Values shown in all graphs represent the average prime editing efficiency of three biological replicates and error bars represent the standard deviation. Data were fitted to four-parameter logistic curves using nonlinear regression.
Extended Data Fig. 2 Optimization of eVLP cargo loading and delivery.
a, A dual transfection/transduction experiment with base editor-delivering BE-eVLPs demonstrates that supplementation of sgRNA does not improve BE-eVLP editing efficiency. b, Adopting the flip-and-extend (F+E) guide RNA scaffold in epegRNAs modestly improves editing efficiencies of v2.3 PE-eVLPs at HEK3 and Dnmt1 in HEK293T and N2A cells respectively. c, Comparison of v2.3 PE-eVLP editing efficiencies at the Dnmt1 locus in N2A cells with Gag–Pol, or a 3:1 ratio of Gag–Pol:Gag–MCP–Pol. d, Comparison of v2.3 PE-eVLPs with one copy or two copies of MCP fused to Gag–Pol. e, Editing efficiencies of v2.3 PE-eVLPs at the Dnmt1 locus in N2A cells with MS2 stem–loop insertions in epegRNA. Zero, one, or two copies of MS2 stem–loop were inserted at various locations of epegRNAs. The position of the MS2 stem–loop insertion is as follows: 3’ denotes v2.3 PE-eVLPs with insertion of the MS2 stem–loop after the structured tevoPreQ1 motif of the epegRNA; 3’* denotes v2.3 PE-eVLPs with insertion of the MS2 stem–loop directly after the 3’-extension of the pegRNA, thereby using the MS2 stem–loop to mimic a structured motif at the 3’ end of the epegRNAs; TL denotes v2.3 PE-eVLPs with insertion of the MS2 stem–loop within the tetraloop of the pegRNA scaffold; ST2 denotes v2.3 PE-eVLPs with insertion of the MS2 stem–loop within the ST2 loop of the pegRNA scaffold. Values shown in all graphs represent the average base editing efficiency (a) or prime editing efficiency (b-e) of three biological replicates and error bars represent the standard deviation. Data were fitted to four-parameter logistic curves using nonlinear regression.
Extended Data Fig. 3 Optimization of eVLP cargo loading and delivery.
a, Fold change in PE-eVLP editing efficiency compared to the original mismatched 5’ G + 20-bp epegRNA protospacer. b, Quantification of the number of eVLP particles per unit volume in preparations of successive generations of PE-eVLPs by anti-MLV p30 ELISA. These quantification data were used in experiments to determine the number of prime editor protein and epegRNA molecules per eVLP shown in Fig. 2g,h. c, Percentage of epegRNA and ngRNA composition in v3 PE2-eVLPs and v3 PE3-eVLPs. Data represent the average value of three technical replicates and error bars represent the standard deviation.
Extended Data Fig. 4 v3b PE-eVLP optimization and characterization.
a, Representative western blot comparing expression of the gag-PE fusion protein from v3 PE-eVLPs versus the P4–PE fusion protein from v3b PE-eVLPs in producer cells transfected with the corresponding fusion proteins. b, Prime editing efficiencies of v3b PE-eVLPs with Gag–P3–Pol or Gag–MCP–P3–Pol. The Gag–MCP–P3–Pol fusion construct is not compatible with the efficient production of PE-eVLPs. c, Editing efficiencies of v3b PE-eVLPs at the Dnmt1 locus in N2A cells with the Com aptamer inserted at various locations in the epegRNAs. The position of the Com aptamer insertion is as follows: 3’ denotes v3b PE-eVLPs with insertion of the Com aptamer after the structured tevoPreQ1 motif of the epegRNA; 3’* denotes v3b PE-eVLPs with insertion of the Com aptamer directly after the 3’-extension of the pegRNA, thereby using the Com aptamer to mimic a structured motif at the 3’ end of epegRNAs; TL denotes v3b PE-eVLPs with insertion of the Com aptamer within the tetraloop of the pegRNA scaffold; ST2 denotes v3b PE-eVLPs with insertion of the Com aptamer within the ST2 loop of the pegRNA scaffold. d, Representative western blot evaluating the amount of PE cargo packaged in v1.3, v2.3, v3 and v3b PE-eVLPs. Figures shown in (a) and (d) are representative images from two independently repeated experiments. Values shown in (b) and (c) represent the average prime editing efficiency of three biological replicates and error bars represent the standard deviation.
Extended Data Fig. 5 Characterization of PE-eVLPs.
a, Comparison of prime editing efficiency (% editing) and insertion-deletion byproduct generation (% indel) of PE3 system delivered by plasmid transfection versus v3 PE3-eVLP. PE3 system targets Dnmt1 locus in N2A cells. Data represent the average prime editing efficiency of three biological replicates and error bars represent the standard deviation. b, Prime editing efficiencies at Dnmt1 locus in N2A cells and HEK3 locus in HEK293T cells from treatment with four independent batches of v3 PE3-eVLPs produced on different days, with each dot indicating the prime editing efficiency of each of the four v3 PE3-eVLP batches. Data shown represent the mean prime editing efficiency from four different v3 PE3-eVLP batches and error bars represent the standard deviation.
Extended Data Fig. 6 Schematic summary of PE-eVLP designs.
Schematic of accessory proteins, cargo proteins, guide RNA designs, and description of improvements from the previous version for successive generations of PE-eVLPs. Envelope protein VSV-G, and capsid protein MMLV Gag–Pol that are common in all versions of PE-eVLPs are omitted from the table. Schematics shown in the table represent PE2-eVLPs. For PE3-eVLPs, additional ngRNAs are packaged at a ratio of 4:1 for pegRNAs and ngRNAs with corresponding scaffold modification and aptamer insertion.
Extended Data Fig. 7 BE-eVLPs benefit from the engineered architectures of v3 and v3b PE-eVLPs.
a-c, Comparison of base editing efficiencies of (a) ABE8e, (b) ABE7.10-NG, and (c) TadCBE at the BCL11A locus in HEK293T cells treated with eVLPs that use the v4 BE-eVLP architecture, the v3 PE-eVLP architecture, or the v3b PE-eVLP architecture. Values shown in all graphs represent the average base editing efficiency of three biological replicates and error bars represent the standard deviation. Data were fitted to four-parameter logistic curves using nonlinear regression.
Extended Data Fig. 8 Example FACS gating for single nucleus sorting.
a, Single nucleus was gated based on forward scatter (FSC-A) and back scatter (BCS-A) ratios and DyeCycle Ruby signal. GFP-positive nuclei were gated based on the FITC signal. The first row displays representative FACS data for untreated samples and the second row displays representative FACS data for cortex samples harvested from neonatal mice co-injected with 4 μl PE-eVLPs and 0.3 μl VSV-G pseudotyped GFP:KASH lentivirus via ICV injection. Bulk nuclei correspond to events that passed gate C and GFP-positive nuclei correspond to events that passed gate D. b, Example FACS gating for neuron-specific sorting. Single nucleus was gated based on forward scatter (FSC-A) and back scatter (BCS-A) ratios and DAPI signal. The signal from Alexa 647-conjugated NeuN antibody distinguishes NeuN-positive and NeuN-negative populations. GFP-positive nuclei were gated based on FITC signal in both NeuN-positive and NeuN-negative populations. Gates displayed represent FACS data for midbrain samples harvested from neonatal mice co-injected with 4 μl PE-eVLPs and 0.3 μl VSV-G pseudotyped GFP:KASH lentivirus via ICV injection. Gate F represents GFP-positive nuclei from the NeuN-negative population. Gate G represents GFP-positive nuclei from the NeuN-positive population.
Extended Data Fig. 9 Immunohistochemistry blot on eye cryosections of rd6 mice.
Retina cryosections from untreated rd6 mice and v3 PE3b-eVLP-treated rd6 mice were stained with DAPI (blue). Figure shown is a representative image from two independently repeated experiments.
Extended Data Fig. 10 Off-target analysis in rd12 mice.
a, Analysis of PE-dependent editing at the on-target site and at the top 10 CIRCLE-seq nominated off-target sites associated with the rd12 epegRNA sequence. b, Analysis of indels at the on-target site and the top 10 CIRCLE-seq nominated off-target sites associated with the rd12 ngRNA sequence. Bars represent average values for n = 3 (untreated) or n = 3 (v3 PE3b-eVLP-treated), with each dot representing an individual mouse and error bars representing standard deviation.
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An, M., Raguram, A., Du, S.W. et al. Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo.
Nat Biotechnol (2024). https://doi.org/10.1038/s41587-023-02078-y
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Received: 18 June 2023
Accepted: 30 November 2023
Published: 08 January 2024
DOI: https://doi.org/10.1038/s41587-023-02078-y
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