Development of an ultra-efficient prime editing system in tomato

Domain: biology
Published: 2026-01-12
Editorial outcome: major_revision

Abstract
Prime editing (PE) enables precise genome modifications without donor DNA or double-strand breaks, but its application in dicot plants has faced challenges due to low efficiency, locus dependence, and poor heritability. Here, we develop an ultra-efficient prime editing (UtPE) system for dicots by integrating evolved PE6 variants (PE6c and PE6ec), an altered pegRNA (aepegRNA), an RNA chaperone, and a geminiviral replicon. UtPE significantly improves editing performance in tomatoes, with UtPEv1 excelling in simple edits (unstructured RTTs) and UtPEv3 effective for complex targets (structured RTTs or multiple nucleotide changes). Compared to a PE2max-based tool, UtPE increases desired editing efficiency by 3.39 to 8.89-fold, enables editing at previously inaccessible sites, achieves an average of 16.0% desired editing efficiency in calli, and produces high-frequency desired edits in up to 87.5% of T0 plants. Multiplexed editing at up to three loci and stable T1 inheritance are also achieved, resulting in traits such as jointless pedicels and glyphosate resistance, while minimizing off-target effects.

Introduction
Prime editing (PE) technology1 enables the precise installation of desired edits at genomic sites in monocots with high efficiency2–20. PE applications in dicot plants faced more challenges than in monocots, as various attempts resulted in very low efficiency, and no heritable PE event was observed21–25. Recently, we developed a dicot PE system that also enabled efficient and heritable PE in dicots, such as tomato and Arabidopsis26. Around the same time, the PE tool was also applied to engineering source-sink relations in tomato27, Arabidopsis28,29, and tobacco29. However, the PE tools have still mainly been locus-dependent and demanded substantial improvement for routine applications for dicot breeding. In our previous work, only six out of eleven sites in tomato exhibited moderate to high desired PE efficiency, and heritable PE events were not achieved at some targets, such as SlHKT1;2, SlEPSPS1, SlCENH3 (sub), and SlDMR626. Therefore, we sought to develop a more efficient and locus-independent PE system for PE-inactive sites, thereby enabling the routine application of PE tools in dicots.

Recently, Liu’s group developed PE6 variants based on the evolution of the PEmax tool and a Tf1 reverse transcriptase (RT), which significantly improved PE efficiency in mammalian cells30. The PE6c and PE6d variants have been shown to be more efficient than the PEmax system in rice using T-DNA with Agrobacterium-mediated transformation (AMT)31,32. However, none of the PE6 variants have been adapted in dicots. Moreover, there were various PE6 variants such as PE6e, PE6f, PE6g, and combinations of the evolved nCas9 and Tf1 (PE6ec, PE6fc, and PE6gc)30, which showed different PE characteristics and capacities in human cells, but those variants have not been evaluated in plants.

In our previous work, we harnessed the PEmax protein, engineered it with an altered engineered PE guide RNA (aepegRNA) system26,33, and enhanced it with a nucleocapsid (NC) RNA chaperone14. Together, these components delivered by a geminiviral replicon vector broke the PE efficiency barrier in tomato and Arabidopsis. We hypothesize that a hyper-efficient and locus-independent PE system should inherit all the best and updated features of the current PE components.

In this work, we integrate and evaluate the most efficient and potential PE6 variants, such as PE6c and PE6ec, with or without the NC chaperone, within our recently developed dicot PE system (Fig. 1a), at multiple tomato targets. Ultimately, we develop an ultra-efficient PE (UtPE) system to improve desired PE efficiency. The UtPE system works very efficiently at all the tested PE-inactive sites and may thus enable locus-independent PE in tomato and beyond.

Results
Integrating the PE6 protein variants into the dicot PE system
All the PE6 variants, their derivatives, and engineered PE proteins with the C-terminal fusion of a nucleocapsid (NC) RNA chaperone peptide tested in this study were co-expressed with an aepegRNA cassette and delivered by a geminiviral replicon system (Fig. 1a and b, and Supplementary Fig. 1). The aepegRNA scaffold contains a stabilized small hairpin34 and the optimized ‘flip and extension’ (F+E) tetraloop35, and it is protected by a trimmed evopreQ (tevopreQ1) loop33 at its 3’ end (Fig. 1b). The transcription of aepegRNA was driven by a U6 composite promoter (pU6cm), a pol II-pol III fusion promoter36, and a double terminator (EURb7). The EuRb7 terminator is an enhanced terminator that contains a genomic insulator (Rb7), which strongly enhanced gene expression (>160-fold increase compared to an opaline synthase (NOS) terminator) and was further enhanced when combined with a geminiviral replicon37. The double terminator was also used to regulate the transcription of the PE6 protein variants together with a long CaMV promoter (Fig. 1a and b, and Supplementary Fig. 1). The PE system, when delivered by an EHA105-based super Agrobacterium version 2 (EHA105spv2)-mediated transformation of the geminiviral replicon, plays a critical role in boosting pegRNA transcription and PE protein production26 and ultimately constitutes an ultra PE (UtPE) system (Fig. 1a).

UtPEv1 outperformed PE6d and the previous dicot PE system in tomato
Recently, PE6c and PE6d variants were adopted in rice with various performances for base substitutions31 and in-locus protein tagging32. Therefore, we initially integrated the PE6c and PE6d protein variants (Fig. 1b) to precisely insert nine nucleotides (5’-AAATTGTGA-3’) at the SlCAB13 site (Supplementary Table 1) that exhibited moderate desired PE efficiency in our recent study26. Analyzing callus explants by targeted deep sequencing, we found that the PE6c variant outperformed the PE2max and PE6d variants (Fig. 1c). The highest average desired PE efficiency reached 11.74 ± 1.74% using PE6c compared to 2.95 ± 0.57% for PE2max, representing a 3.98-fold enhancement (Fig. 1c and Supplementary Table 2). PE6c slightly increased the desired/imprecise edit ratios and did not increase indel mutations compared to PE2max (Fig. 1c and Supplementary Table 2), similar to the observation in human cells30. Meanwhile, the PE6d variant was just slightly better than PE2max with a 1.29-fold improvement.

The processivity of RT enzymes may be enhanced by an RNA chaperone that facilitates the destabilization of stem loops formed by a complexed RTT14. Indeed, we previously employed the NC RNA chaperone to enhance PE activity by fusing it to the C-terminus of PE2max in tomato and Arabidopsis26. Unexpectedly, when we added NC to the C-terminal of PE6c, the PE efficiency was only comparable to that of PE6c alone (Fig. 1c and Supplementary Table 2). In contrast, adding NC at the C-terminal of PE6d significantly improved its performance (Fig. 1c and Supplementary Table 2). The PE6c variant also outperformed PE2max-NC with 1.35-fold higher desired PE efficiency, reaching up to 16.03 ± 1.34% at the SlCAB13 site (Fig. 1d and Supplementary Table 3). The system using the PE6c and PE6c-NC variants is designated as UtPEv1 and UtPEv2, respectively (Fig. 1a).

To further validate the UtPEv1 activity, we employed UtPEv1 to install the desired edits at six additional sites (Supplementary Tables 1 and 4) that had previously shown nil to moderate PE activity26. In the callus stage, we found that UtPEv1 outperformed the previous PE2max-based dicot PE system at all tested sites, with a 3.39- to 8.89-fold increase (Fig. 2a and Supplementary Table 4). Interestingly, though the PE2max-based dicot PE system did not work at the SlDMR6 site, UtPEv1 exhibited 8.09 ± 0.25% desired PE efficiency, albeit with high imprecise PE (mostly scaffold inserted) and indel mutation rates (Supplementary Table 4). The desired/imprecise edit ratio of UtPEv1 was only reduced at inactive sites, such as SlCENH3 (del) and SlHKT1;2, compared to PE2max. At moderate to highly active sites, UtPEv1 even slightly improved the desired/imprecise edit ratio while the indel mutation rate was comparable (Supplementary Table 4). Among the sites, the desired PE efficiency reached an average of 14.86 ± 0.47%. Moreover, except for the SlHKT1;2 site, all the others resulted in more than 2% of the desired PE efficiency at the callus stage (Fig. 2a), a practical level for obtaining PE events carrying the desired edits at high frequencies.

UtPEv3 improved desired PE efficiency at the sites with more complex RTTs
The desired edit to be installed at the SlHKT1;2 site is complex with three desired nucleotide changes (+10 A to G; +19 A to G; and +21 T to C), including synonymous mutations (Supplementary Table 1), forming a predicted stem loop within its RTT strand (Supplementary Fig. 2) and may require a more robust PE variant for installing it. The PE6c and PE6d were developed based on the combinations of nCas9 from PEmax and evolved Tf1 RT and MMLV RT, respectively30. While PE6c and PE6d were well characterized in human cells and recently adapted to monocots31,32, the evolved nCas9 configurations of PE6e-g, which showed potential PE improvement in human cells, have not yet been tested elsewhere. In this experiment, we engineered and tested PE6ec (combined evolved nCas9 of PE6e and evolved Tf1 RT of PE6c), PE6fc (combined evolved nCas9 of PE6f and evolved Tf1 RT of PE6c), and PE6gc (combined evolved nCas9 of PE6g and evolved Tf1 RT of PE6c) (Fig. 1b) for improving PE efficiency at the SlHKT1;2 site. However, none of the combinations could enhance the activity of the PE tools as UtPEv1 still resulted in the highest desired PE efficiency at the target site (callus stage) (Fig. 2b). Except for the PE6ec variant, all the other combinations of the evolved nCas9s and Tf1 RT resulted in a dramatic reduction of PE performance compared to UtPEv1 (Fig. 2b). Simultaneously, we fused the RNA chaperone NC to the C-terminal of PE6ec, PE6fc, and PE6gc variants (Fig. 1b) and assessed its impacts on their activity. The addition of NC could enhance the desired efficiency of PE6ec, resulting in a 1.37-fold increase over UtPEv1. Similarly, UtPEv2 was slightly better (1.26-fold increase) than UtPEv1 (Fig. 2a and Supplementary Table 5). The NC chaperone also led to more imprecise edits as the desired/imprecise edit ratios reduced in the cases of UtPEv2 and PE6ec-NC (Supplementary Table 5). Interestingly, the PE6ec-NC variant showed the highest desired PE efficiency, 1.76 ± 0.23%, at this site. It is designated as UtPEv3 (Fig. 1a).

Since the UtPEv1, UtPEv2, and UtPEv3 exhibited comparable desired PE efficiency at the SlHKT1;2 site, we assessed their activities at three more targets (SlOR, SlCENH3 (sub), and SlCAB13) and found that the desired PE efficiency was almost comparable among the UtPE tools at the callus stage (Fig. 2c). The desired PE efficiency was highest with UtPEv1 at SlOR (9.81 ± 0.76%) and SlCAB13 (14.68 ± 1.14%) and with UtPEv2 at SlCENH3 (sub) (1.94 ± 0.53%) site (Fig. 2c and Supplementary Table 6). Moreover, the UtPE variants exhibited similar desired/imprecise edit ratios (Fig. 2d) and indel mutation rates (Supplementary Tables 5 and 6) compared to the PE2max-based dicot PE system at the four tested sites.

The UtPE system shows low gRNA-dependent off-target activity
Since the activity of the UtPE variants is significantly higher than that of PE2max, we wonder if they could induce higher pegRNA-dependent off-target activity. We employed the Cas-OFFinder38 for identifying potential off-target sites of the gRNAs in tomato (Supplementary Data 1). Assessing two potential off-target sites per pegRNA revealed no off-target traces of PE editing and only background levels of indel mutation frequency from the UtPE and PE2max systems (Fig. 2e and f), as also observed earlier26.

The tRNA-HDV-based UtPE outperformed the Csy4-aided UtPE
The Csy4 protein (also known as Cas6f) is the key enzyme responsible for crRNA production in CRISPR subtype I-F39. Csy4 bound with equal affinity to both its pre-crRNA substrate and Csy4 stem-containing 3’ end of the processed crRNA39, raising the possibility of 3’ end protection by Csy4 protein39,40 (Fig. 3a). The Csy4-processing system was employed to support pegRNA and sgRNA production in human cells41,42, wheat43, rice40, and tomato27. We compared the Csy4-aided aepegRNA processing system with the tRNA-HDV-based aepegRNA processing (Fig. 1a) UtPE tool by replacing the tRNA-HDV sequences with the Csy4 recognition sequence (Csy4RS) and co-expressed Csy4 protein together with the PE tool (Fig. 3b). The Csy4-UtPEv1-containing construct has a slightly higher size (~2.3 kb) than the UtPEv1-delivering replicon thanks to the addition of the expression cassette of Csy4 protein (Fig. 3b). Unexpectedly, our data revealed that the Csy4-assissted UtPEv1 system showed significantly lower PE efficiency (0.4-0.86-fold lower) compared to the UtPEv1 tool at all three tested sites (Fig. 3c and Supplementary Table 7).

The UtPE tool enables efficient multiplexing PE in tomato
Efficient and simultaneous editing of multiple targets at high precision is highly desirable for engineering traits that are linked to various genes/alleles44. Our previous version of the dicot PE tool was shown to be feasible for multiplexed PE, with slightly reduced PE efficiencies compared to single-PE tools26. We tested whether the UtPE tool could also enable multiplexed PE. UtPEv1 and UtPEv3 were employed to simultaneously install desired edits at two (SlOR and SlCAB13) and three (SlOR, SlCAB13, and SlPRD) targets (Fig. 3b). Via NGS, we found that the UtPEv1 and UtPEv3 tool showed comparable desired PE efficiencies with the single UtPE construct and UtPEv3 was slightly better than UtPEv1 in multiplexed PE with three targets (Fig. 3d and Supplementary Table 7).

The UtPE system efficiently produced the desired PE events at all the tested sites
Screening T0 transformant revealed potential events carrying desired PE edits from all the targets with the UtPE tools, with 5/8 sites resulting in events carrying homozygous desired edits (Table 1, Supplementary Figs. 3 and 4). The desired PE efficiency of the UtPE tools at the plant stage varied between 2.9 and 87.5%, depending on the targets, and is much higher compared with PE2max and PE2max-NC (Table 1). Interestingly, UtPEv3 exhibited the highest desired PE efficiency levels at the sites with longer and multiple edit-containing RTTs such as SlCAB13 (87.5%) and SlHKT1;2 (6.5%), while UtPEv1 and UtPEv2 resulted in more events without containing scaffold-inserted alleles for the sites with simpler RTTs like SlOR (33.3%) and SlCENH3 (sub) (5.9%), respectively (Supplementary Figs. 3 and 4, and Table 1). We could then validate most of the representative PE events by targeted deep sequencing (Supplementary Table 8). At the SlHKT1;2 target, while no event was obtained with UtPEv2 and two potential events with UtPEv3 (Supplementary Fig. 3 and Table 1), only potential events generated by UtPEv3 could be confirmed to carry a high desired PE frequency (Supplementary Table 8). This result may be due to the low desired edit frequency (less than 15%) of both the potential events obtained with UtPEv1, whereas those obtained with UtPEv3 showed a ~40% desired edit frequency in the ICE Synthego screening.

Subsequently, in our screening for transgenes, we identified two out of sixteen representative T0 events that are likely free of replicon and show very low T-DNA amplification (Supplementary Fig. 5). These data indicate that the developed UtPE system is hyper-efficient and could enable locus-independent PE in tomatoes. We recommend using UtPEv1 for targets with simple RTTs and UtPEv3 for sites with long and complex RTTs.

The desired PE alleles installed by the UtPE system were stably inherited and caused phenotypic changes
It is essential to verify that the desired PE edits installed by the UtPE system are heritable and produce the desired phenotypic changes. We, therefore, assessed the inheritance of the desired PE edits in the next generation of the PE events generated by the UtPE tools. We identified the desired PE alleles in T1 plants from representative T0 events, in both homozygous and heterozygous forms (Supplementary Table 9). We found that plants with chimeric scaffold-inserted alleles or indels did not pass these alleles to the next generation, indicating they were likely temporary changes. In contrast, T0 plants with a mix of scaffold-inserted or indel alleles did pass these alleles to the T1 generation, following Mendel’s laws of inheritance. Among the homozygous T1 plants, we observed a relatively high frequency of plants lacking T-DNA and replicon amplification (18 out of 42 analyzed plants) (Supplementary Fig. 6). The T1 plants carrying homozygous KO allele pairs of SlMBP21 exhibited the jointless phenotype (Fig. 4a), as reported earlier45. In the case of plants carrying the homozygous SlEPSPS1 (TIPS) allele, 5 mM glyphosate resistance was observed (Fig. 4b). However, it appears that the TIPS protein variant has weaker catalytic activity in the shikimate pathway46, leading to growth retardation in the plants. Further enhancement of SlEPSPS1 (TIPS) expression by promoter engineering may result in glyphosate tolerance and normal growth as observed in overexpressing EPSPS1 (TIPS) driven by a strong promoter in rice47 and cassava48. Assessing the haploid induction efficiency of the SlCENH3 (del) and SlCENH3 (sub) alleles involved crossing T1 plants that carried homozygous forms of these alleles with hairless/trichomeless plants (Supplementary Fig. 7a). This approach revealed some potential haploid F1 progenies that exhibited reduced hair on their stems (Supplementary Fig. 7b). However, upon sequencing the SlCENH3 gene, it became evident that all potential F1 progenies were either chimeric or heterozygous (Supplementary Fig. 7c and Supplementary Table 10). This finding indicates that haploid induction in these progenies was either unsuccessful or incomplete.

Methods
System design and plasmid cloning
The PE2max and PE2max-NC variants were cloned from a plasmid of our published work26 (Addgene plasmid #227693). The PE6c and PE6d variants were cloned according to the published work of Doman and coworkers30 using Addgene plasmids (#207853 and #207854, respectively). The PE6ec, PE6fc, and PE6gc were designed and cloned by combining nCas9 from PE6e (Addgene plasmid #207855), PE6f (Addgene plasmid #207856), and PE6g (Addgene plasmid #207857), respectively, with the evolved Tf1 RT (evoTf1: P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, K118R, S188K, I260L, S297Q, R288Q) of the PE6c plasmid (see Supplementary Data 2). The RNA chaperone NC (from pH-ePPE, Addgene plasmid #183097) was fused to the C-terminal of PE6 variants to generate PE6c-NC, PE6d-NC, PE6ec-NC, PE6fc-NC, and PE6gc-NC (Fig. 1a and b and Supplementary Data 2). The PE protein expression cassettes were driven by the CaMV 35S promoter (Addgene plasmid #50267) and EURb7 terminator (Fig. 1a and Supplementary Data 2)26. All the pegRNAs were previously designed and cloned, with an aepegRNA configuration that was used with the pU6cm by Vu and coworkers26 (Fig. 1b and Supplementary Fig. 1 and Supplementary Table 1). The Csy4-aepegRNAs were cloned by replacing the tRNA(gly) and HDV sequences of the aepegRNAs with Csy4RS sequences (Fig. 3a and Supplementary Data 2). The Csy4 expression cassette was driven by the CaMV 35S promoter (Addgene plasmid #50267) and NOS terminator (Addgene plasmid #50339) (Fig. 3b and Supplementary Data 2). All the PE constructs were cloned into a geminiviral replicon vector system (Addgene plasmid #227697)26,50 for plant transformation with a selection marker (NptII, Addgene #51144) (Supplementary Fig. 1). All cloning work was performed using the Golden Gate assembly method53. All bioparts (Supplementary Data 2) were incorporated into the Moclo level 0 plasmids and assembled into the PE protein combinations and binary vectors, as shown in Fig. 1a and b, and Supplementary Fig. 1.

Tomato transformation
The PE tools were transformed into tomato cotyledon slices by Agrobacterium (super Agrobacterium version 2 strain (EHA105spv2))-mediated transformation protocol reported earlier by Vu and coworkers26. The tomato MoneyMaker (MM) variety was used in this study. In detail, cotyledons that were 7 days old were excised for transformation. Agrobacterium containing the PE constructs was cultured, harvested, and resuspended in a modified ABM-MS medium. This medium consisted of ABM (composed of 2.21 g/L K2HPO4, 1.13 g/L KH2PO4, 1.5 g/L NH4NO3, 0.15 g/L KCl, 0.31 g/L MgSO4, 0.015 g/L CaCl2, 0.003 g/L FeSO4, 3.9 g/L MES, and 20 g/L glucose at pH 5.5) mixed in a 1:1 ratio with 1/4 MSB5 (which includes MS basal salts with Gamborg B5 vitamins from Duchefa Biochemie B.V, Haarlem, Netherlands, Cat. M0231, along with 5 g/L sucrose, pH 5.2). The optical density (OD600nm) of the Agrobacterium suspension was adjusted to 0.8, and 200 µM acetosyringone (Sigma-Aldrich Inc., Darmstadt, Germany, Cat. D134406) was added. The Agrobacterium cells were then activated at 28°C for 1 h in a shaker set to 180 rpm. After this activation, the Agrobacterium cells were mixed with the cotyledon explants and incubated at room temperature for 20 min. The transformed explants were then placed on co-cultivation medium, which consisted of semisolid ABM-MS containing 1 mg/L zeatin trans-isomer (Duchefa Biochemie B.V, Haarlem, Netherlands, Cat. Z0917), 1 mM putrescine dihydrochloride (Sigma-Aldrich Inc., Darmstadt, Germany, Cat. P5780), 200 µM acetosyringone, and 7.5 g/L agar. They were incubated at 25°C for 2 days before being washed and transferred to a selection medium. This selection medium contained full MSB5, 1.0 mg/L zeatin trans-isomer, 0.1 mg/L 3-Indoleacetic acid (Sigma-Aldrich Inc., Darmstadt, Germany, Cat. I2886), 1 mM putrescine dihydrochloride, 30 g/L glucose, 300 mg/L timentin, and 80 mg/L kanamycin, with a pH of 5.8. Explants were then incubated at 34°C for 5 days and subsequently maintained at 28°C for an additional 5 days before being shifted to 25°C for the remaining stages (T34 incubation regime). The conditions were previously shown to result in the maximal activity of the PE2max tool by Vu et al.26. Regenerated shoots were rooted in rooting medium ((haft MSB5, 1 mg/L Indole-3-butyric acid (Sigma-Aldrich Inc., Darmstadt, Germany, Cat. 57310), and 30 g/L glucose, 300 mg/L timentin, and 80 mg/L kanamycin, pH 5.8), and then transferred to soil.

Targeted deep sequencing
To assess PE efficiency in the callus stage, samples were collected at 16 days post-transformation (dpt) and analyzed via targeted deep sequencing. Targeted deep sequencing was performed as previously reported26. Briefly, genomic DNA was extracted from cotyledon explants or plant leaves using the GENTi™ Advanced Genomic DNA Extraction Kit and the GeneAll® GENTi™ 32 Advanced automated nucleic acid extraction system (Jinol Biotechnology Co., Ltd., Seoul, Korea) using the manufacturer’s protocol. Targeted deep sequencing was performed using the Illumina MiniSeq platform (KAIST Bio-Core Center). Libraries were prepared via three rounds of PCR, with sample indexing in the third round using manufacturer-supplied primers (see Supplementary Data 3). FASTQ files were analyzed using CRISPResso254.

Screening T0 transformants and T1 plants carrying PE alleles
T0 transformants that survived the hardening step and T1 offspring plants from T0 events were screened for PE alleles. Three leaf fragments were collected from three different compound leaves of each plant and combined to isolate gDNA using the GENTi™ Advanced Genomic DNA Extraction Kit and the GeneAll® GENTi™ 32 Advanced automated nucleic acid extraction system (Jinol Biotechnology Co., Ltd., Seoul, Korea) using the manufacturer’s protocol. PCR reactions were used to amplify DNA sequences using primers (Supplementary Data 3) that flank the targeted sites, and the PCR products were sequenced by the Sanger method. The resulting sequencing chromatograms were analyzed using the Synthego ICE Analysis tool (version 3)55 to screen for events carrying the desired PE alleles. Some representative PE events were subsequently validated by targeted deep sequencing. In Table 1, The number of desired PE events was calculated by subtracting the number of plants containing scaffold-inserted alleles from the total number of plants with the desired PE.
‘Total number of analyzed plants’ might be smaller than the sum of ‘Number of plants with desired PE’ ‘Number of plants contained indel’, and ‘Number of plants contained WT allele’, since an analyzed plant may contain both desired PE edits and imprecise edits or indels.
The desired PE efficiency was calculated using the following equation:
Desired PE efficiency (%) = 100 × (number of desired PE events / total number of analyzed plants) (1).

Assessment of T-DNA and replicon presence within PE events
The replicon cassette is part of the T-DNA but can still replicate briefly after the T-DNA is removed or modified50,51. This allows us to detect replicon DNA even if the original T-DNA sequences are missing. To address this, we created a dual genotyping method to distinguish between stable T-DNA integration and the persistence of episomal replicons. The presence of T-DNA and replicon within the PE events was assessed by PCR26. In general, PCR reactions using primer pairs specified for the right border (RB) of the T-DNA and the circularized forms of the replicon (Supplementary Data 3) were conducted with SlGAPDH (Solyc05g014470) as an internal control. PCR products were separated on a 1% agarose gel, and the absence of T-DNA and replicon was determined by the lack of bands corresponding to their expected sizes without having any other unspecific bands. Each experiment was performed twice and yielded similar results.

Potential off-target analysis
The potential off-target analysis was conducted similarly to a previous report by Vu et al.26. In brief, the gRNA sequences of the pegRNAs (see Supplementary Table 1) were analyzed using the Cas-OFFinder38 web-based application to identify potential off-target sites in the tomato (Solanum lycopersicum) genome database. This search was conducted using the assembly variant SL2.40, accessed in March 2024 at https://solgenomics.net/. Only off-target sites that had fewer than four mismatches to the gRNA sequences were considered in this analysis. Two potential off-target sites per target gRNA/pegRNA were selected for the assessment (Supplementary Data 1). The 16-dpt cotyledon explants that were transformed with pegRNAs having identified potential off-target sites were subjected to analysis by targeted deep sequencing using primers listed in Supplementary Data 3. Targeted deep sequencing was performed using the Illumina MiniSeq platform (KAIST Bio-Core Center), and FASTQ files were analyzed using the CRISPResso2 program.

Herbicide resistance assay
Eight-leaf stage tomato seedlings carrying homozygous SlEPSPS1 (TIPS) and WT were sprayed two times (a five-minute interval) with 5 mM glyphosate isopropylamine (Farmhannong, Seoul, Korea) solution containing Silwett L-77 (0.02%). Sprayed plants were photographed after one and two weeks post-spraying.

Assessment of haploid induction by the SlCENH3 (del) and SlCENH3 (sub) alleles
Due to the lack of flow cytometry and karyotyping facilities for ploidy assessment, we employed an alternative method to assess the haploid induction caused by the tomato SlCENH3 (del) and SlCENH3 (sub) alleles. T1 plants carrying a homozygous form of the desired edits (upper stem containing hairs/trichomes) were cross-pollinated with plants carrying a homozygous knockout form (a recessive allele) of the HAIRS ABSENT gene56, which are hairless. The resulting first filial generation (F1) plants were screened for the presence/absence of hairs and sequenced to identify the SlCENH3 allelic forms. Normal fertilization results in plants carrying heterozygous alleles (SlCENH3/SlCENH3(del) or SlCENH3/SlCENH3(sub)) and hairs, while male haploid plants induced by SlCENH3 mutations should possess the wild type (WT) CENH3 allele with a hairless phenotype (Supplementary Fig. 7a).

Data analysis and presentation
All the experiments were carried out at least in triplicate. Editing data, as well as any applicable statistical analyses and scatter plots, was subsequently processed using Microsoft Excel 365 and the GraphPad Prism 9.0 software. Multiple comparisons were executed utilizing the two-sided, uncorrected Fisher’s LSD test. Figures 1a and b and 3a, as well as Supplementary Fig. 7a were prepared using Biorender (biorender.com) under a paid subscription.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Authors developed prime editors efficient in dicots based on PE6c variants and their previously described expression strategies. This study is timely and interesting and provides better prime editing tools for dicots.\nMajor concerns:\nSince Csy4-aided prime editors have been reported are efficient in dicots (Lou, H. et al. Cell, 2025: 188, 530-549.e520), I am eager to know whether or not UtPEs described in this report can be enhanced by the Csy4 system.\nMinor revisions\n1. What are simple edits and complex targets in Abstract？These terms should be clearly indicated.\n2. What is the standard for “high-frequency” in 87.5% of T0 plants showing high-frequency desired edits? It should be indicated.\n3. Sanger sequencing chromatograms in Fig 3 represent limited information, which is suggested to move to Supplemental Information.\n4. Please indicate the calculation method for the Desired PE efficiency in Table 1.\n5. Please check the values for SlOR/UtPEv2 in Table 1.", "reviewer_2": "* General comments:\nPrime Editing (PE) tools have attracted considerable attention for their ability to perform a variety of genetic modifications. but their use in dicotyledonous plants such as tomato and Arabidopsis is limited by their low efficiency. To address this issue, several groups, including the Kim's research team, have developed advanced PE versions for dicots with high efficiency. In the present study, Vu et al. demonstrated an ultra-efficient prime editing (UtPE) system, which adopted PE6 protein variants (PE6c and PE6ec) with an altered engineered prime editing guide RNA (aepegRNA), a nucleocapsid (NC) RNA chaperone, and a geminiviral replicon for delivery. Overall, the experiment was well designed and the manuscript was easy to read.\nGiven the importance and interest in PE applications to dicotyledons, it might be a good fit for this journal; however, the reviewer raises several concerns including low novelty.\n* Specific comments:\n1. One of the main concerns of this study is its originality and novelty compared to previous studies even conducted by the same group [References #26, #33]. As I understand it, this study combines previous studies of #26 (using aepegRNA, NC RNA chaperone, and geminivirus replicon) and of #33 (using PE6 mutations). If I missed any other key points of this study, please highlight them in the Discussion section.\n(minor)\n- The details of “imprecise edits” need to be clarified. Did they include insertion and deletion mutations?\n- It might be better to move the “off-target analysis data” in the main figure.\n- Please use the abbreviation \"PE\" after it was defined.", "reviewer_3": "The manuscript by Vu et al. provides an incremental, yet positive improvement of a previous research from the same group aimed at increasing the efficiency of prime editing (PE) in plants, particularly focused on the vegetable crop model tomato.\nThe manuscript is well written and although it has a very technical language, it is relatively easy to understand.\nHowever, the manuscript does not seem to offer a unifying solution for Prime Editing in tomato (or dicots) and instead shows that efficiency of desired edits varies among genes and different types of UtPEv# or PE versions. Their improvements seem to be coming from their previous work and rather add more complexity to a system that is already challenged by the amount of modifications needed to work. Furthermore, the authors basically show the same target genes with a modified system, which narrows down even more the scope of their results. Additionally, their genetic analysis is confusing and lacks enough representation to confirm their claim that the edits are stably inherited or to at least demonstrate that there are no segregation distortions from the function of the PE system or the complex transgene used for expressing it.\nHere are few more specific comments:\nThe data on Table 1 seems confusing or inconsistent. The total shown on column “Total no. of analyzed plants” does not add up to the numbers shown in total column of No. of plants with desired PE. If I add up the numbers in columns 8, 9 & 10, I don’t get the total number of plants analyzed (Column 4). Even worse, the % efficiency of PE in the last column does not correspond to the % of column 8. Finally, Where are the WT or unedited plants from this screening? Were all 30 plants analyzed containing edits? If I take the values of columns 8, 9 & 10, I get 32, not 30 from the SlOR data. It seems the data in SlOR is inaccurate and is not clear where the % PE efficiency from the last column is coming from.\nLine 113 seems to refer to the wrong table (S1) in the supplemental materials.\nFigure S7 data seems to be coming from just a few individuals, and the transgene segregation seems off. The number of T1 plants shown for each gene is too small to make any meaningful assessment of segregation of the transgene and the PE modifications. Under the assumption of independent segregation between the transgene and the target genes, I’d expect a 1:2:1 T-DNA segregation in the homozygous individuals. So, 3/4 of the shown homozygous samples should be transgenic. Yet, it seems most of the plants are non-transgenic. Even worse, many of the non-transgenic plants exhibit the presence of the replicon. How’s that possible if the replicon is encoded in the transgene itself?\nWhat’s the rationale for genotyping for both the replicon and the T-DNA? Based on figure S1, the whole UtPE and the replicon elements seem to be contained within the T-DNA and the replicon, so wouldn’t you expect that at least plants showing the replicon would also show the transgene band?\nIf the delivery of these vectors is done through stable transformation as described in the methods, then why is it that there’s no correlation between the presence of the T-DNA and the replicon?\nIt is not clear to this reviewer which (or whether) heterozygous or chimeric plants carry scaffold-inserted alleles or indels. Is it common to recover these alleles in T1 or subsequent generations? Or are these more spurious events that may not be relevant? It would be worth discussing this in the results or discussion sections.\nThe authors targeted 6 genes, many of them from their previous publication. The authors showed that some of the UtPEV systems are better than others at delivering PE desired edits. However, they only showed phenotypes for 2 of the target genes. Given that PE is very promising on delivering precise editing, it seems a bit narrow on scope to choose genes for which loss-of-function mutations are needed to express a phenotype (MBP21, DMR6, CENH3). It would have been more significant to engineer gain-of-function or modify cis-regulatory mutations to demonstrate that PE offers a significant breakthrough compared to more conventional approaches such as base editing or even SDN-1 type of mutations.\nFurthermore, it would have been more interesting to see if PE can be used as a multiplexing tool. Even using these 6 genes as a test case. If PE is to be considered a valuable tool for breeding, it would be necessary to show it’s efficient at delivering complex editing at more than one site at a time.\nAdditionally, the authors don’t show the other phenotypes for target genes CAB13, CENH3, OR or DMR6. It would be very important to include that data, as it’s known that cenh3 mutants may exhibit fertility defects, or dmr6 mutants exhibit reduced stature and reduced fruit size or weight. And those phenotypes would help showing the heritability and phenotypic impact of PE mutations in the target traits."}, "author_response": "Our response: Thank you very much for taking the time to read our manuscript and provide valuable comments and suggestions. We are doing our best to improve the manuscript based on your insightful feedback.\nMajor concerns:\nSince Csy4-aided prime editors have been reported are efficient in dicots (Lou, H. et al. Cell, 2025: 188, 530-549.e520), I am eager to know whether or not UtPEs described in this report can be enhanced by the Csy4 system.\nOur response: Thank you for this constructive suggestion. We have now updated the data with the Csy4-aided UtPE in Fig. 3 of the revised manuscript. Interestingly, the Csy4-aided UtPEv1 tool appeared to negatively affect the PE efficiency at all three tested targets. As previously reported, high doses or overaccumulation of Csy4 protein have been shown to cause cytotoxicity in human cells (Nissim et al., 2014) and to severely affect Agrobacterium-mediated transformation in maize and Arabidopsis (Jiang et al., 2020). Further, Jiang and coworkers (2020) could not obtain any transgenic maize plants with the Csy4-aided system. In our study, the UtPE tool employs the geminiviral replicon which facilitates enhanced expression of the pegRNA and PE protein as shown earlier in our work (Vu et al., 2024). We hypothesized that this enhanced expression system also leads to an overaccumulation of the Csy4 protein, leading to cell death and reduced PE efficiency. This may explain the difference between our results and the findings of Lou et al., given the high-expression nature of our geminiviral replicon system. Indeed, we experimentally observed slower growth and a less healthy status for transformed cotyledon explants treated with the Csy4-aided PE compared to the UtPE tool.\nReferences:\nNissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell. 2014 May 22;54(4):698-710. doi: 10.1016/j.molcel.2014.04.022.\nJiang, Y. Y., Y. P. Chai, M. H. Lu, et al. 2020. “Prime Editing Efficiently Generates W542L and S621I Double Mutations in Two ALS Genes in Maize.” Genome Biology 21: 257.\nVu TV, Nguyen NT, Kim J, Song YJ, Nguyen TH, Kim JY. Optimized dicot prime editing enables heritable desired edits in tomato and Arabidopsis. Nat Plants. 2024 Oct;10(10):1502-1513. doi: 10.1038/s41477-024-01786-w.\nMinor revisions\n1. What are simple edits and complex targets in Abstract？These terms should be clearly indicated.\nOur response: Thank you for the comment. We have now revised the text.\n2. What is the standard for “high-frequency” in 87.5% of T0 plants showing high-frequency desired edits? It should be indicated.\nOur response: We have now clarified the standard for “high-frequency” in the manuscript. A T0 plant was defined as having 'high-frequency' edits if the desired PE edit frequency was >10%, which aligns with our classification criteria for Chimeric (10-44%), Heterozygous (45-94%), or Homozygous (95-100%) events (as detailed in Table 1 footnotes). In the case of the SlCAB13 target, 87.5% (28/32) of T0 plants met this >10% standard. Among these 28 high-frequency events, the majority (24/28) were further classified as either heterozygous or homozygous (Table 1).\n2\n3. Sanger sequencing chromatograms in Fig 3 represent limited information, which is suggested to move to Supplemental Information.\nOur response: Thank you for the suggestion. We have now moved the figure to Supplemental Information as Fig. S3\n4. Please indicate the calculation method for the Desired PE efficiency in Table 1.\nOur response: Thank you for the suggestion. The calculation method has now been added. To make it clearer, we added the column ‘No. of desired PE events’. ‘No. of desired PE events’= ‘Total no. of plants with desired PE’ – ‘No. of events contained scaffold-inserted alleles’. The desired PE efficiency is equal to 100*(No. of desired PE events/total number of analyzed plants).\n5. Please check the values for SlOR/UtPEv2 in Table 1.\nOur response: We apologize for the confusion regarding the values for SlOR/UtPEv2. We have thoroughly reexamined the dataset and corrected the table.\nThe column ‘No. of desired PE events’ is calculated as follows: ‘Total number of plants with desired PE’ minus ‘No. of plants containing scaffold-inserted alleles.’ Please also note that an analyzed plant may contain both desired PE edits and imprecise edits or indels. Thus, ‘Total number of analyzed plants’ might be smaller than the sum of ‘No. of plants with desired PE,’ ‘No. of plants containing indels’, and ‘No. of plants containing WT alleles.’\nFor the UtPEv2 at the SlOR targets, we found one T0 plant that carries a high frequency of desired edits and a low frequency of scaffold-inserted nucleotides. Initially, we considered this to represent a potential desired PE event, but in the revised version, we excluded this event from the ‘No. of desired PE events’ for clarity.\nReviewer #2 (Remarks to the Author):\n* General comments:\nPrime Editing (PE) tools have attracted considerable attention for their ability to perform a variety of genetic modifications. but their use in dicotyledonous plants such as tomato and Arabidopsis is limited by their low efficiency. To address this issue, several groups, including the Kim's research team, have developed advanced PE versions for dicots with high efficiency. In the present study, Vu et al. demonstrated an ultra-efficient prime editing (UtPE) system, which adopted PE6 protein variants (PE6c and PE6ec) with an altered engineered prime editing guide RNA (aepegRNA), a nucleocapsid (NC) RNA chaperone, and a geminiviral replicon for delivery. Overall, the experiment was well designed and the manuscript was easy to read.\nGiven the importance and interest in PE applications to dicotyledons, it might be a good fit for this journal; however, the reviewer raises several concerns including low novelty.\nOur response: We sincerely appreciate your time and effort in reviewing our manuscript and providing thoughtful comments and suggestions. Your valuable feedback is greatly helping us to refine and enhance the quality of our work.\n* Specific comments:\n1. One of the main concerns of this study is its originality and novelty compared to previous studies even conducted by the same group [References #26, #33]. As I understand it, this study combines previous studies of #26 (using aepegRNA, NC RNA chaperone, and geminivirus replicon) and of #33 (using PE6 mutations). If I missed any other key points of this study, please highlight them in the Discussion section.\nOur response: We sincerely thank the reviewer for this valuable comment. While this study indeed builds upon our previous work (Ref #26) and other (Ref. #33), it presents several original and substantial advances:\n1. This report presents the first systematic adaptation and evaluation of the evolved PE6 variants (PE6c, PE6d, PE6ec, PE6fc, and PE6gc) and their fusion with the NC RNA chaperone in dicot plants. Notably, the performance of the PE6ec-NC fusion using the UtPEv3 tool has not been previously documented in either animals or plants.\n2. We developed an integrated ultra-efficient platform by combining PE6 variants with our optimized dicot-specific components: aepegRNA, the NC RNA chaperone, and a geminiviral replicon. This new system, called UtPE, significantly outperforms previous dicot PE tools in terms of efficiency, locus independence, and heritability.\n4\n3. Demonstration of locus-independent and heritable prime editing: The UtPE system successfully performed efficient editing at previously uneditable loci, such as SlHKT1;2, SlDMR6, and SlCENH3. It generated up to 87.5% heritable edits in T0 plants, which has not been previously reported in dicots.\n4. Efficient multiplexed PE (updated data): UtPE allowed efficient simultaneous editing of up to three targets, achieving comparable or higher efficiency than single-target editing.\n5. We differentiated UtPEv1 (PE6c-based) and UtPEv3 (PE6ec+NC-based) based on RTT complexity, offering practical guidance for selecting tools in future applications.\nTo clarify these points, we have revised the Discussion section to explicitly highlight these advances and distinguish the UtPE system from previous studies (added lines #252-259 in the revised manuscript).\n(minor)\n- The details of “imprecise edits” need to be clarified. Did they include insertion and deletion mutations?\nOur response: Thank you for the point. The “imprecise edits” is the sum of “unintentionally modified prime edited portion” and “prime edited portion with scaffold insertion”. We added the clarification in all the related Figure legends. The imprecise edits include scaffold insertion and short sequence deletion and insertion beside the prime edits. The other insertion and deletion that are independent of the prime editing events are shown as indel mutations.\n- It might be better to move the “off-target analysis data” in the main figure.\nOur response: Thank you. We have now prepared a Figure panel (Fig. 2E-F) for the potential off-target analysis data.\n- Please use the abbreviation \"PE\" after it was defined.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Vu et al. provides an incremental, yet positive improvement of a previous research from the same group aimed at increasing the efficiency of prime editing (PE) in plants, particularly focused on the vegetable crop model tomato. The manuscript is well written and although it has a very technical language, it is relatively easy to understand.\nOur response: We sincerely appreciate the time and effort you dedicated to reviewing our manuscript and for your encouraging remarks. We are making every effort to revise the manuscript in line with your helpful comments and suggestions.\nHowever, the manuscript does not seem to offer a unifying solution for Prime Editing in tomato (or dicots) and instead shows that efficiency of desired edits varies among genes and different types of UtPEv# or PE versions. Their improvements seem to be coming from their previous work and rather add more complexity to a system that is already challenged by the amount of modifications needed to work. Furthermore, the authors basically show the same target genes with a modified system, which narrows down even more the scope of their results.\nOur response: We appreciate the reviewer’s insightful assessment. Since prime editing efficiency was locus- and context-dependent in dicots, as shown in our previous study (Vu et al, 2024), we sought to overcome the limitations by upgrading the previous system to a more robust UtPE system. In the case, we think the best way is to directly compare the previous system with the newly engineered PE tools at the tested targets, especially those that could not be edited by the previous PE variants.\nThis work is the first systematic adaptation and evaluation of the evolved PE6 variants (PE6c, PE6d, PE6ec, PE6fc, and PE6gc) and their fusion with the NC RNA chaperone in dicot plants. Notably, the performance of the PE6ec-NC fusion using the UtPEv3 tool has not been previously documented in either animals or plants.\nIn addition, our aim was not to propose a single “universal” system, but to establish a robust, modular platform (UtPEv1–v3) that integrates key performance-enhancing components and\n6\nallows flexible optimization across diverse loci.\nTo clarify these points, we have added more discussion (lines #252-262).\nAdditionally, their genetic analysis is confusing and lacks enough representation to confirm their claim that the edits are stably inherited or to at least demonstrate that there are no segregation distortions from the function of the PE system or the complex transgene used for expressing it.\nOur response: We appreciate the reviewer's concern regarding the stability of the edits. We have generated and performed genotypic assessment of T0 events and T1 segregants. To further address this, we have updated the data to include T1 segregants from 23 independent T0 events across 08 target sites (Fig. S7). Our analysis confirms that T0 plants carrying heterozygous desired edits transmitted these alleles to the T1 generation following Mendelian inheritance patterns, demonstrating stable inheritance and indicating no significant segregation distortion caused by the PE system or the complex transgene.\nHere are few more specific comments:\nThe data on Table 1 seems confusing or inconsistent. The total shown on column “Total no. of analyzed plants” does not add up to the numbers shown in total column of No. of plants with desired PE. If I add up the numbers in columns 8, 9 & 10, I don’t get the total number of plants analyzed (Column 4). Even worse, the % efficiency of PE in the last column does not correspond to the % of column 8. Finally, Where are the WT or unedited plants from this screening? Were all 30 plants analyzed containing edits? If I take the values of columns 8, 9 & 10, I get 32, not 30 from the SlOR data. It seems the data in SlOR is inaccurate and is not clear where the % PE efficiency from the last column is coming from.\nOur response: We thank the reviewer for noticing this and apologize for any confusion it might cause.\nWe have thoroughly reexamined the dataset and added two new columns titled ‘No. of plants containing WT allele’ and ‘No. of desired PE events.’ The column ‘No. of desired PE events’ is calculated as follows: ‘Total number of plants with desired PE’ minus ‘No. of plants containing scaffold-inserted alleles.’ Please also note that an analyzed plant may contain both desired PE edits and imprecise edits or indels. Thus, ‘Total number of analyzed plants’ might be smaller\n7\nthan the sum of ‘No. of plants with desired PE,’ ‘No. of plants containing indels’, and ‘No. of plants containing WT alleles.’\nFor the UtPEv2 at the SlOR targets, we found one T0 plant that carries a high frequency of desired edits and a low frequency of scaffold-inserted nucleotides. Initially, we considered this to represent a potential desired PE event, but in the revised version, we excluded this event from the ‘No. of desired PE events’ for clarity.\nAccordingly, we have corrected the numbers in the revised Table 1. The desired PE efficiency is calculated using the formula: 100 * (No. of desired PE events / total number of analyzed plants). These corrections are now reflected in the revised version of Table 1, its legend, and in the Methods section (lines #367-372).\nLine 113 seems to refer to the wrong table (S1) in the supplemental materials.\nOur response: Thank you for bringing this to our attention. This reference is to introduce the target sites, their desired edits, and pegRNA components. We have now updated the reference to “Table S1 and S4” in the revised manuscript (line #115).\nFigure S7 data seems to be coming from just a few individuals, and the transgene segregation seems off. The number of T1 plants shown for each gene is too small to make any meaningful assessment of segregation of the transgene and the PE modifications. Under the assumption of independent segregation between the transgene and the target genes, I’d expect a 1:2:1 T-DNA segregation in the homozygous individuals. So, 3/4 of the shown homozygous samples should be transgenic. Yet, it seems most of the plants are non-transgenic. Even worse, many of the non-transgenic plants exhibit the presence of the replicon. How’s that possible if the replicon is encoded in the transgene itself?\nOur response: We acknowledge that the T1 segregation dataset represents a limited number of individuals due to the technical challenge of genotyping both transgene and PE alleles in multiple loci simultaneously. We believe that the detection and validation of the desired edits in a number of T1 plants derived from various T0 independent events, encompassing up to eight target sites, is sufficient to demonstrate the stable inheritance of these edits in the T1 generation. In the future, we will focus on analyzing the genetic segregation of the desired edits from multiple loci. We have now updated the data with T1 segregants from additional T0 events and target sites.\nRegarding the apparent detection of the replicon in non-transgenic plants, we noted that the geminiviral replicon can transiently persist as an episome during early regeneration stages, even\n8\nafter loss of integrated T-DNA (Cermak et al., 2015; Vu et al., 2020), which explains the occasional replicon signal in T-DNA–negative plants. Moreover, T-DNA integration was shown unnecessary for achieving genome edited plants (Cermak et al., 2015). Cermak and cowrkers suggested that T-DNA merely functions as a delivery vehicle for the viral replicons, and that its integration is not required. We have added this explanation to the Discussion section (lines 278-287) with a citation to previous reports describing similar episomal maintenance behavior (Cermak et al., 2015; Vu et al., 2020).\nReferences\nČermák T, Baltes NJ, Čegan R. et al. High-frequency, precise modification of the tomato genome. Genome Biol 16, 232 (2015). https://doi.org/10.1186/s13059-015-0796-9.\nVu TV, Sivankalyani V, Kim EJ, et al. Highly efficient homology-directed repair using CRISPR/Cpf1-geminiviral replicon in tomato. Plant Biotechnol J. 2020 Oct;18(10):2133-2143. doi: 10.1111/pbi.13373.\nWhat’s the rationale for genotyping for both the replicon and the T-DNA? Based on figure S1, the whole UtPE and the replicon elements seem to be contained within the T-DNA and the replicon, so wouldn’t you expect that at least plants showing the replicon would also show the transgene band?\nIf the delivery of these vectors is done through stable transformation as described in the methods, then why is it that there’s no correlation between the presence of the T-DNA and the replicon?\nOur response: We acknowledge that the initial version may not have made this point clear. While the replicon cassette is encoded within the T-DNA, it can continue to replicate autonomously for a period of time even after T-DNA is degraded. This can result in the appearance of replicon DNA even when intact T-DNA sequences are absent. Similar observations were reported in previous studies on geminiviral replicons (Cermak et al., 2015; Vu et al., 2020). Consequently, we designed the dual genotyping to differentiate between stable T-DNA integration and the persistence of episomal replicons. This rationale has been clarified in the revised Methods section (lines #374-377).\nOur response: We appreciate this insightful question. We observed that plants carry chimeric scaffold-inserted alleles or indels did not transmitted the chimeric alleles to the next generation, suggesting they represent transient or somatic events rather than stable germline modifications. The T0 plants that carried heterozygous form of scaffold-inserted or indel alleles stably transmitted the alleles into T1 generations and they seem to follow Mendelian segregation rule. We have added several sentences in the Results section (lines #216-219) clarifying this observation.\nThe authors targeted 6 genes, many of them from their previous publication. The authors showed that some of the UtPEV systems are better than others at delivering PE desired edits. However, they only showed phenotypes for 2 of the target genes. Given that PE is very promising on delivering precise editing, it seems a bit narrow on scope to choose genes for which loss-of-function mutations are needed to express a phenotype (MBP21, DMR6, CENH3). It would have been more significant to engineer gain-of-function or modify cis-regulatory mutations to demonstrate that PE offers a significant breakthrough compared to more conventional approaches such as base editing or even SDN-1 type of mutations.\nOur response: We appreciate this comment. In deed, in this work we targeted 8 genes with 9 pegRNAs and majority (7/9) are to install gain-of-function edits (Table S1). In our previous work (Vu et al., 2024), we have phenotyped the desired edits of SlALS1 (herbicide tolerance) and SlOR (enhanced carotenoid production). The KO allele of SlMBP21 and SlDMR6 were precisely generated by introducing stop codon at the target sites differing from pre-mature stop codon generated via indel mutations by the targeted mutagenesis approach. The SlCENH3 (sub) and SlCENH3(del) allele are both gain-of-function and they require precisely changes of a.a and 6-nt deletion, respectively.\nWe focused phenotypic validation on SlMBP21 and SlEPSPS1 because these loci provided visible phenotypes to showcase the expression of the desired edits in the T1 plants. We have now\n10\nupdated data regarding the phenotypic assessment of SlCENH3(sub) and SlCENH3(del) alleles (lines #228-234, 293-302, 398-408 and Fig. S9).\nIn the future, we will have a more thorough phenotypic analysis of all the remaining desired edits.\nWe agree that future studies aimed at generating cis-regulatory or gain-of-function edits will further expand the scope of PE applications, as now noted in the revised Discussion (lines 302-307).\nFurthermore, it would have been more interesting to see if PE can be used as a multiplexing tool. Even using these 6 genes as a test case. If PE is to be considered a valuable tool for breeding, it would be necessary to show it’s efficient at delivering complex editing at more than one site at a time.\nOur response: We agree that multiplexing represents an important direction for practical PE application. In the revised manuscript, we have included multiplexed PE data (new data added in Fig. 3D), showing that simultaneous edits at three loci were achievable with moderate efficiency. This result is now described in revised text (lines #34-35, 179-188 and #245-246).\nAdditionally, the authors don’t show the other phenotypes for target genes CAB13, CENH3, OR or DMR6. It would be very important to include that data, as it’s known that cenh3 mutants may exhibit fertility defects, or dmr6 mutants exhibit reduced stature and reduced fruit size or weight. And those phenotypes would help showing the heritability and phenotypic impact of PE mutations in the target traits.\nOur response: Thank you for the point. In our previous work (Vu et al., 2024), we have phenotyped the desired edits of SlOR (enhanced carotenoid production). We have now updated data regarding the phenotypic assessment of SlCENH3(sub) and SlCENH3(del) alleles (lines #228-234, 293-302, 398-408 and Fig. S9). The SlCAB13 or SlDMR6 desired edits require more complex system or biotic stress tolerance facilities, respectively, to be assessed so we would have collaboration to the lab that has can test the phenotypes in the near future.\n11"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Please check the annotation of Csy4 expression cassette.\nNo other comments.", "reviewer_2": "​Although, given previous research by the same group, concerns remain about its originality and novelty, I agree with the publication of this revised version.", "reviewer_3": "- Many thanks to the authors for providing a thorough response to this reviewer's comments. My only still standing major concern related to the authors rebutal of their genotyping data showing contrasting results between the presence of the T-DNA and the replicons (episomes?).\n- The authors cited Cermak et al., 2015 as a reference to indicate that some edited plants may be transgene-free and still contain replicons. However, Cermak et al specifically found that replicons only persists up to 8 weeks post-inoculation, which is not even the full-cycle of tomato growth (accounting for tissue culture and greenhouse growth). Cermak et al performed PCR and western blots showing that gene-targeted mature plants lacked replicons. This is text from their original paper:\n\"No evidence of circular replicons was observed in any of the 26 mature plants recovered from events 1, 2, 10 and 11 using the first primer pair (Fig. 4b). Similarly, PCR performed with the other primer pair did not detect the presence of T-DNA or replicons in any of the five T0 plants tested (one from each GT event) or 34 T1 progeny\"\n\"..replicons were detected in the transformed tissue up to 8 weeks post-inoculation, but not in mature plants\"\n- Therefore, it seems unlikely for this reviewer that the authors could detect replicons in mature T0 plants or T1 progeny that are transgene-free. I do not understand how this could be possible, unless the replicons are acting as a seed-transmissible virus. Perhaps it would be worth indicating at which stage these DNA samples were collected (tissue culture stage, young T0 or mature T0). That still wouldn't explain the results from figure S8 (T1 segregation).\nMinor comments:\n- Figure S3 appears such low-res it's really hard to make sense of it."}, "author_response": "Author's response to Reviewers' comments\nReviewer #1 (Remarks to the Author):\nPlease check the annotation of Csy4 expression cassette.\nOur response: Thank you for pointing out it. We have now revised the annotation of Csy4 expression cassette in the legend of Fig. 3 and the Supplementary sequences.\nReviewer #2 (Remarks to the Author):\nAlthough, given previous research by the same group, concerns remain about its originality and novelty, I agree with the publication of this revised version.\nOur response: Thank you for the agreement.\nReviewer #3 (Remarks to the Author):\n- Many thanks to the authors for providing a thorough response to this reviewer's comments.\nMy only still standing major concern related to the authors rebutal of their genotyping data showing contrasting results between the presence of the T-DNA and the replicons (episomes?).\nOur response: Thank you for your important comments and suggestions to further improve our manuscript. Our explanation for the remaining concern is shown below.\n- The authors cited Cermak et al., 2015 as a reference to indicate that some edited plants may be transgene-free and still contain replicons. However, Cermak et al specifically found that replicons only persists up to 8 weeks post-inoculation, which is not even the full-cycle of tomato growth (accounting for tissue culture and greenhouse growth). Cermak et al performed PCR and western blots showing that gene-targeted mature plants lacked replicons. This is text from their original paper:\n1\n\"No evidence of circular replicons was observed in any of the 26 mature plants recovered from events 1, 2, 10 and 11 using the first primer pair (Fig. 4b). Similarly, PCR performed with the other primer pair did not detect the presence of T-DNA or replicons in any of the five T0 plants tested (one from each GT event) or 34 T1 progeny\"\n\"..replicons were detected in the transformed tissue up to 8 weeks post-inoculation, but not in mature plants\"\n- Therefore, it seems unlikely for this reviewer that the authors could detect replicons in mature T0 plants or T1 progeny that are transgene-free. I do not understand how this could be possible, unless the replicons are acting as a seed-transmissible virus. Perhaps it would be worth indicating at which stage these DNA samples were collected (tissue culture stage, young T0 or mature T0). That still wouldn't explain the results from figure S8 (T1 segregation).\nOur response: Thank you for raising this critical point. We appreciate the opportunity to clarify the structural differences between our system and the one reported by Cermak et al. (2015).\nWhile Cermak et al. reported the loss of replicons in mature plants, this outcome was likely attributed to their specific construct design. In their study (and our previous work using a similar design; Vu et al., 2020), the geminiviral replicon contained a CaMV 35S promoter within the donor template driving a selection marker, placed in reverse orientation to the replicon’s LIR promoter. We hypothesize that this convergent transcription from the opposing LIR and CaMV 35S promoters generated double-stranded RNA, triggering RNAi-mediated silencing and the subsequent clearance of the replicon in mature tissues.\nCrucially, in the same Vu et al. (2020) study, when we used a donor template lacking the opposing CaMV 35S promoter (for SlHKT1;2 editing), we successfully detected replicons in both mature T0 and T1 plants. This strongly suggests that in the absence of RNAi-inducing transcriptional collision, replicons can persist.\nIn the current study, we utilized a system similar to the latter design (without the opposing dual promoters). Consequently, the persistence of replicons in our T0 and T1 plants is consistent with the absence of silencing triggers. Regarding the transmission mechanism, since the viral coat protein gene was deleted, the replicons cannot function as a contagious virus; their presence in T1 likely results from high stability and inheritance during cell division rather than viral infection. We have added this discussion to the revised manuscript (lines #279-288).\nReferences:\nMinor comments:\n- Figure S3 appears such low-res it's really hard to make sense of it.\nOur response: Thank you for pointing out it. We have now revised the Figure S3 to a higher resolution one.\n3"}, {"round": 3, "reviewer_comments": {"reviewer_1": "No further comments.", "reviewer_3": "Thank you for addressing my concerns, I am satisfied with the manuscript modifications."}, "author_response": ""}]

Decision letter