Skip to main content
NCBI home page
BMB Reports logoLink to BMB Reports
. 2025 Jan 13;58(2):70–74. doi: 10.5483/BMBRep.2024-0137

Development of a prime editor with improved editing efficiency in Arabidopsis

Yeong Yeop Jeong 1,2,3,#, Cheljong Hong 2,#, Jun Hee Han 4,#, Sangsu Bae 5,6,7,*, Pil Joon Seo 1,2,3,*
PMCID: PMC11875749  PMID: 39681408

Abstract

Prime editing is widely used in many organisms to introduce site-specific sequence modifications such as base substitutions, insertions, and deletions in genomic DNA without generating double-strand breaks. Despite wide-ranging applications of prime editing, prime editors (PEs) have low editing efficiencies, especially in dicot plants. Therefore, PEs are barely used for genome engineering in dicot plant species. Here, based on previous approaches used to improve prime editing efficiency, we generated different combinations of PE components and prime editing guide RNAs (pegRNAs) and examined their prime editing efficiencies in Arabidopsis thaliana protoplasts as a dicot model system. We found that v4e2, in which PE was fused to viral nucleocapsid (NC) protein, RNase H-deleted M-MLV RT, and a dominant negative version of human mutL homolog 1 (hMLH1dn), showed the highest prime editing efficiency in Arabidopsis protoplasts when it was co-transfected with dual enhanced pegRNA. Our results suggest that the v4e2 PE system could be used for efficient prime editing in dicot plants.

Keywords: Arabidopsis, Dual-pegRNA, Genome editing, Prime editing, Protoplast

INTRODUCTION

Prime editing has been widely used to generate targeted, site-specific, DNA sequence modifications including nucleotide substitutions, insertions, and deletions in a wide range of organisms (1). Prime editors (PEs) are composed of Cas9 nickase (nCas9) containing a H840A mutation fused to Moloney-murine leukemia virus reverse transcriptase (M-MLV RT). To serve as a template for directly editing the target site with reverse transcription, the engineered prime editing guide RNA (pegRNA) contains a primer binding site that marks the initiation site for reverse transcription and a reverse transcription template (RTT) sequence encoding the desired edit (2).

The first generation of PE (PE1) was designed by fusing M-MLV RT to either the N- or C-terminal end of nCas9 (H840A) indispensable for prime editing (2). Since its development, the PE system has been further improved mainly by modifying its protein components. The PE2 system was developed by introducing point mutations (D200N/L603W/T330P/T306K/W313F) in M-MLV RT that could improve its thermostability and cDNA synthesis efficiency (2). An engineered plant prime editor (ePPE) was also created by removing the RNase H domain of M-MLV RT (∆RNase H) and introducing a viral nucleocapsid (NC) protein (3). Furthermore, based on the finding that mismatch repair (MMR) interferes with prime editing, a dominant negative version of the human mutL homolog 1 protein (hMLH1dn), a component of the MutSα-MutLα MMR complex, was introduced into PE2 or PE3 to decrease undesired byproducts in the edited strand (PE4 or PE5) (4). The fusion of PEs to the RNA-binding N-terminal domain of La, a small RNA-binding exonuclease protection factor, could also improve prime editing (PE7) (5).

Additionally, the use of engineered pegRNA (epegRNAs) can greatly enhance prime editing efficiency (6). Stability and reverse transcription of pegRNAs can be enhanced by adding RNA secondary structures to pegRNA (6) and adjusting length of the primer binding site (7). A dual-pegRNA strategy, in which two separate pegRNAs introduce the same edits in each DNA strand, can also increase prime editing efficiency (7).

PEs have been improved and exploited mainly in monocot plants, including rice, wheat, and maize (8-13). However, their applications in dicot plants remain limited because of their low editing efficiencies in such plants. Here, based on previous strategies used to improve prime editing efficiency, we generated multiple combinations of PE components and pegRNAs and found that the v4e2 variant showed the highest prime editing efficiency in Arabidopsis thaliana, a model dicot, raising the possibility of prime editing in dicot plants.

RESULTS AND DISCUSSION

A protoplast transient expression assay is a versatile, rapid, and high-throughput method for assessing genome editing efficiency (14, 15). To efficiently evaluate editing efficiencies of multiple PE variants, we optimized our protocol for transient expression assays by increasing protoplast yield and viability (16). To isolate protoplasts, two-week-old seedlings (Fig. 1A) were incubated with an enzyme solution at room temperature for 6 h (Fig. 1B) (16). A sucrose density gradient purification method was then employed to enrich viable protoplasts (Fig. 1C-E). Freshly prepared protoplasts were used strictly for polyethylene glycol (PEG)/Ca2＋-mediated transfection of plasmid DNAs. Protoplasts transfected with an AtUBQ10::NLS-mCherry plasmid showed a transfection efficiency of approximately 46% at 18 h after transfection (Fig. 1F, G). An optimized transient expression assay was subsequently used for evaluating editing efficiencies of PE variants.

Fig. 1.

Fig. 1

Open in a new tab

Preparation and transfection of Arabidopsis protoplasts. (A) Two-week-old seedlings grown under long-day (16 h light/8 h dark) conditions. Scale bar, 1 cm. (B) Protoplast isolation using an enzyme solution. Scale bar, 1 cm. (C) Protoplast solution dispensed over 0.6 M sucrose solution for density gradient-based protoplast purification. Scale bar, 1 cm. (D) Photograph showing a dark-green band containing intact protoplasts that formed at the interface of 0.6 M sucrose solution and MMC (10 mM MES, 0.47 M Mannitol, 10 mM Calcium) solution after centrifugation. Scale bar, 1 cm. (E) Photograph of purified protoplasts captured under a microscope. Scale bar, 100 μm. (F) Images of protoplasts expressing NLS-mCherry. Protoplasts were incubated at 23°C in the dark and fluorescence images were captured at 18 h post-transfection using a CQ1 confocal quantitative image cytometer. Fluorescence of FDA is shown in green and that of mCherry is shown in red. Scale bar, 100 μm. (G) Ratio of NLS-mCherry expressing protoplasts to viable protoplasts. Transfection efficiency was quantified using a CQ1 confocal quantitative image cytometer. Each dot represents a biological replicate. Data are presented as mean ± SEM (standard error of the mean) of three independent biological replicates. P-value was obtained using two-tailed Student’s t-test.

Previous studies have shown that prime editing efficiency can be improved by modifying PE components and/or engineering pegRNAs (2-6). Exploiting these strategies, we generated eight PE variants in this study using varying combinations of Arabidopsis codon-optimized nCas9 (R221K/N394K/H840A) (4), tomato codon-optimized 34-amino acid flexible linker as well as M-MLV RT (8), ∆RNase H (M-MLV lacking the RNase H domain) (3), dominant negative human MLH1 protein (hMLH1dn) (4), viral NC with nucleic acid chaperone activity (3), engineered pegRNA (epegRNA, generated by incorporating structured RNA motifs into the 3’ end of pegRNA) (6), and dual-pegRNAs (7) (Fig. 2A). We used 2×35S and AtU6 promoters to express PE variants and pegRNAs, respectively. Two different genomic regions (AtGL1 and AtPDS3) were chosen as target loci for editing to create a stop codon at the leucine 87 (L87) position of the AtGL1 protein and to disrupt the splicing acceptor site of the first intron of AtPDS3 (Fig. 2B).

Fig. 2.

Fig. 2

Open in a new tab

Prime editing in transiently transformed Arabidopsis protoplasts. (A) Schematic representation of eight PE variants. 35S, 35S promoter; tNOS, NOS terminator. (B) Details of target sites (AtGL1 and AtPDS3) used for assessing editing efficiencies of different prime editors (PEs). Genomic sites targeted for prime editing are underlined. STOP, premature stop codon. (C) Assessment of eight PEs at two different target sites in Arabidopsis protoplasts. Each dot represents a biological replicate. Data are presented as mean ± SEM. Different letters represent significant differences at P < 0.05 (one-way ANOVA with Tukey’s post hoc test). (D) Analysis of editing outcomes at AtGL1 and AtPDS3 loci using v4e2 variants. Seven editing patterns with the highest read counts are displayed and v4e2-mediated nucleotide polymorphisms are highlighted in orange. WT, wild-type; PE, prime editing; Sub, substitution.

To identify PE variant exhibiting the highest prime editing efficiency in the model system, we transiently expressed each PE variant construct into Arabidopsis protoplasts for three days. Genomic DNAs were then isolated from transfected protoplasts for targeted deep sequencing. Although prime editing efficiency varied in the context of sequence, v4e2 showed the highest editing frequency at target sites among all PE variants examined (Fig. 2C). Notably, the relative position of nCas9 and M-MLV RT influenced editing efficiency (2, 17). A v4e2 variant, in which M-MLV RT was fused to the C-terminus of nCas9, showed 2.4-fold and 11-fold higher efficiencies of desired editing at AtGL1 and AtPDS3, respectively, than v4Re2, in which M-MLV RT was fused to the N-terminus of nCas9. Furthermore, the dual-epegRNA strategy substantially enhanced prime editing efficiency. Transfection with v4e2 resulted in 10.9-fold and 5.5-fold higher desired editing efficiencies at AtGL1 and AtPDS3, respectively, than that with v4e1. Results of analyzing product purity of v4e2 showed that desired editing was more predominant than imprecise editing (Fig. 2D).

To determine whether our finding could be applied to plant systems, we transformed each AtGL1-targeting PE variant into Arabidopsis plants using a floral dip method (18) to generate stable transgenic plants. Genomic DNAs were then extracted from rosette leaves of T1 plants for high-throughput sequencing analysis. Consistent with results of protoplast transfections assays, v4e2 showed the highest prime editing efficiency (up to 9.11%) in terms of average editing frequency in transgenic Arabidopsis plants (Fig. 3). Product purity of v4e2 in transgenic plants also appeared to be similar to that in Arabidopsis protoplasts. Although we did not try to obtain T2-edited plants, it is highly likely that genome editing is inheritable in transgenic plants, albeit with a low editing efficiency, as observed in previous studies (19-21).

Fig. 3.

Fig. 3

Open in a new tab

Prime editing in stably transformed Arabidopsis plants. (A) Assessment of editing efficiencies of eight PE variants in transgenic Arabidopsis plants in T1 generation. N/A, not available. (B) Analysis of editing outcomes in a transgenic line AtGL1-v4e2 #30. Seven editing patterns with the highest read counts are displayed and v4e2-mediated nucleotide polymorphisms are highlighted in orange. WT, wild-type; PE, prime editing; Sub, substitution.

Overall, v4e2 was found to be the best PE among those tested in Arabidopsis. The v4e2 system could be used for prime editing in other dicot plants, although its editing efficiency is still low. Considering that differences in DNA repair mechanisms between mammals and plants may lead to their variable prime editing efficiencies (20, 22), we speculate that dicot plants likely have molecular processes different from those of monocot plants. Such differences can interfere with prime editing. It is important to elucidate dicot-specific genetic factors that hinder the action of PEs and to engineer PE systems that can bypass genetic hindrance in the future for optimizing prime editing in dicot plants.

MATERIALS AND METHODS

Plasmid construction

All DNA fragments for plasmid construction were amplified with Primestar GXL DNA polymerase (Takara Bio, R050A) and cloned using the Gibson assembly method. The epegRNA scaffolds were designed based on a previous study (6), and dual-epegRNAs were designed using the PlantPegDesigner webtool (6, 7). All pegRNAs were commercially synthesized by Bionics, and all plasmids were confirmed by Sanger sequencing (Bionics). PE-expressing plasmids were constructed using pPPED (backbone plasmid) (Addgene #162468). A variant of nCas9 (R221K/N394K/H840A) was generated by PCR-based site-directed mutagenesis using a primer containing the desired mutation. Arabidopsis codon-optimized MLH1dn was commercially synthesized (Bionics) and cloned into pPPED by adding 2A-cleavage sequences and NLS, resulting in 2×35S::NLS-nCas9 (R221K/N394K/H840A)-linker-M-MLV RT-NLS-P2A-MLH1dn-NLS (v2e1, expressing a single epegRNA). To investigate the effect of the relative order of nCas9 and M-MLV RT on editing efficiency, the 2×35S::NLS-M-MLV RT-linker-nCas9 (R221K/N394K/H840A)-NLS-P2A-MLH1dn-NLS plasmid (v2Re1) was constructed. To construct the v3 variants, including 2×35S::NLS-nCas9 (R221K/N394K/H840A)-linker-viral NC-NLS-linker-M-MLV RT (∆RNase H)-NLS (v3e2, expressing dual-epegRNA) and 2×35S::NLS- M-MLV RT (∆RNase H)-linker-viral NC-NLS-linker-nCas9 (R221K/N394K/H840A)-NLS (v3Re2), the viral NC domain commercially synthesized by Bionics and M-MLV RT deleted for ribonuclease H (∆RNase H) were cloned into the pPPED backbone using Gibson assembly. The v4 variants containing the single epegRNA-expressing cassette including 2×35S::NLS-nCas9 (R221K/N394K/H840A)-linker-viral NC-NLS-linker-M-MLV RT (∆RNase H)-NLS-P2A-MLH1dn-NLS (v4e1) and 2×35S::NLS- M-MLV RT (∆RNase H)-linker-viral NC-NLS-linker-nCas9 (R221K/N394K/H840A)-NLS-P2A-MLH1dn-NLS (v4Re1) were generated by combinational reconstruction. Subsequently, the v4 variants, namely, v4e2 and v4Re2, were constructed by replacing the single epegRNA-expressing cassette with the dual-epegRNA-expressing cassette in v4e1 and v4Re1, respectively.

Plant growth conditions and plant transformation

Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used to perform all experiments. Surface-sterilized seeds were sown on half-strength Murashige and Skoog (MS) medium containing vitamins supplemented with 0.05% (w/v) 2-morpholinoethanesulfonic acid monohydrate (MES·H2O), 1% (w/v) sucrose, and 0.8% (w/v) plant agar. After sowing, the seeds were stratified for 3 days at 4°C in the dark. Seedlings were grown under long-day (16 h light/8 h dark) conditions using white fluorescent lamps (120 μE m−2 s−1) at 23°C.

Arabidopsis plants were transformed with Agrobacterium tumefaciens strain GV3101 harboring individual AtGL1-targeting PE variants using the floral dip method (18) to generate transgenic (T1) lines.

Plasmid DNA preparation

A single colony of DH5α carrying a certain plasmid was picked and grown overnight in a test-tube containing 2 ml of LB medium supplemented with 100 mg/L kanamycin on an orbital shaker (250 rpm) at 37°C. Then, 0.5-ml of the cell suspension was used to inoculate 40 ml of fresh TB medium supplemented with 100 mg/L kanamycin and grown in 250-ml baffled flasks at 37°C for 16 h with shaking (250 rpm). The overnight culture was transferred to a 50-ml tube. Bacterial cells were collected by centrifugation at 6,500 × g for 10 min and resuspended in 4 ml of TEG buffer (25 mM Tris-HCl, 10 mM EDTA, and 50 mM glucose [pH 8.0]). Then, 8 ml of cell lysis solution (0.2 N NaOH and 1.0% (w/v) SDS) was added to the bacterial cell suspension, mixed gently by inverting 5-7 times, and incubated at room temperature for 10 min. The cell lysate was neutralized with 6 ml of potassium acetate (KOAc, 3 M) solution containing 11.5% (v/v) acetic acid and incubated at room temperature for 10 min. After centrifugation at 14,000 × g for 10 min, the supernatant was passed through two layers of Miracloth (Millipore, 475855). The filtrate was collected in a new 50-ml tube, mixed with 10 ml of isopropyl alcohol, and incubated at room temperature for 10 min. Plasmid DNA was collected by centrifugation at 14,000 × g for 10 min. The DNA pellet was washed with 70% (v/v) ethanol, resuspended in 0.6 ml of TE buffer (25 mM Tris-HCl and 10 mM EDTA [pH 8.0]), and transferred into a 2-ml tube. To remove bacterial RNA, the suspension was mixed with 300 μl of 4.2 M CaCl2 solution and incubated at room temperature for 5 min. The tube was centrifuged at 12,000 × g for 10 min. The supernatant was transferred into a new 2-ml tube and mixed with 70 μl of 3 M NaOAc and 0.5 ml of isopropyl alcohol. After incubation at room temperature for 10 min, the tube was centrifugated at 12,000 × g for 10 min. The pellet was washed twice with 70% (v/v) ethanol and resuspended in 0.5 ml of TE buffer. To selectively precipitate the plasmid DNA, an equal volume of PEG-NaCl solution (20% [w/v] PEG 8000 and 0.5 M NaCl) was added to the plasmid DNA and mixed gently by inverting 5-7 times. Immediately after mixing, the tube was centrifugated at 12,000 × g for 10 min. The pellet containing purified plasmid DNA was washed twice with 70% (v/v) ethanol and resuspended in TE buffer. Plasmid DNA concentrations were determined using BioDrop (Biochrom, BD1776) and adjusted to a concentration of 2 μg/μl.

Protoplast preparation

Protoplast isolation and purification were performed as described previously (16), with slight modifications. Briefly, 2-week-old Arabidopsis seedlings were soaked in 20 ml of 0.5 M mannitol solution (pH 5.8) at 23°C for 1 h. The plasmolyzed seedlings were immersed in 20 ml of enzyme solution containing 2% (v/v) Viscozyme L, 1% (v/v) Celluclast 1.5 L, and 1% (v/v) Pectinex ultra SP-L and incubated on a rotating shaker (50-60 rpm) for 6 h. Intact protoplasts were collected using sucrose density gradient centrifugation.

Protoplast transfection assay

Protoplast transfection was performed as described previously (14), with the following modification. Purified protoplasts were resuspended in MMG solution (0.4 M mannitol, 15 mM MgCl2 and 4 mM MES [pH 5.7]) at a concentration of 4 × 106 protoplasts/ml. Twenty micrograms of plasmid DNA was mixed with 300 μl of protoplasts. Then, an equal volume (300 μl) of PEG solution (40% [w/v] PEG 4000, 0.1 M CaCl2, and 0.2 M mannitol) was added to the protoplasts, mixed, and incubated for 5 min at room temperature. After incubation, 1 ml of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, and 5 mM glucose [pH 5.7]) was added to the protoplasts and gently mixed. Protoplasts were collected by centrifugation at 80 × g for 5 min, washed with 1 ml of W5 solution, centrifuged again at 50 × g for 5 min, and resuspended in 100 μl of PIM medium (Gamborg B5 medium containing vitamins, 20 g/L sucrose, 60 g/L myo-inositol, 2 mg/L 6-BAP, and 0.5 mg/L α-NAA [pH 5.8]). The transfected protoplasts were incubated at 23°C in the dark.

To evaluate transfection efficiency, protoplasts were transfected with pCMU-NUCr (Addgene #61168) (23), incubated for 18 h, and then stained with fluorescein diacetate (FDA) (24). The viability and transfection efficiency of protoplasts were assessed using a CQ1 confocal quantitative image cytometer (Yokogawa).

To assess prime editing efficiency, the transfected protoplasts were incubated for 3 days. Subsequently, the protoplasts were collected and subjected to genomic DNA extraction using the cetyltrimethylammonium bromide (CTAB) method for high-throughput sequencing.

High-throughput sequencing

To perform targeted amplicon sequencing, genomic regions containing the target sites were PCR-amplified using primers, including adapters that are compatible with Illumina index barcodes. Amplified DNAs were purified using Expin PCR SV mini (GeneAll, 103-102), and purified libraries were then sequenced using the Miniseq sequencing system (Illumina). After sequencing, paired-end data were analyzed using a PE-analyzer (http://www.rgenome.net/pe-analyzer/) (25). Sequencing data were mostly analyzed using the computing server at the Genomic Medicine Institute Research Service Center.

ACKNOWLEDGEMENTS

This work was supported by the Basic Science Research (NRF-2022R1A2B5B02001266 to P.J.S.; NRF-2021M3A9H3015389 to S.B.) and Basic Research Laboratory (NRF2022R1A4A3024451) programs of the National Research Foundation of Korea, and the New Breeding Technologies Development Program (RS-2024-00322275 to P.J.S.) of the Rural Development Administration.

Footnotes

CONFLICTS OF INTEREST

The authors have no conflicting interests.

REFERENCES

  • 1.Chen PJ, Liu DR. Prime editing for precise and highly versatile genome manipulation. Nat Rev Genet. 2023;24:161–177. doi: 10.1038/s41576-022-00541-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Anzalone AV, Randolph PB, Davis JR, et al. Search- and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576:149–157. doi: 10.1038/s41586-019-1711-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Zong Y, Liu Y, Xue C, et al. An engineered prime editor with enhanced editing efficiency in plants. Nat Biotechnol. 2022;40:1394–1402. doi: 10.1038/s41587-022-01254-w. [DOI] [PubMed] [Google Scholar]
  • 4.Chen PJ, Hussmann JA, Yan J, et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. 2021;184:5635–5652.e5629. doi: 10.1016/j.cell.2021.09.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Yan J, Oyler-Castrillo P, Ravisankar P, et al. Improving prime editing with an endogenous small RNA-binding protein. Nature. 2024;628:639–647. doi: 10.1038/s41586-024-07259-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Nelson JW, Randolph PB, Shen SP, et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol. 2022;40:402–410. doi: 10.1038/s41587-021-01039-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Lin Q, Jin S, Zong Y, et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat Biotechnol. 2021;39:923–927. doi: 10.1038/s41587-021-00868-w. [DOI] [PubMed] [Google Scholar]
  • 8.Wang L, Kaya HB, Zhang N, et al. Spelling changes and fluorescent tagging with prime editing vectors for plants. Front Genome Ed. 2021;3:617553. doi: 10.3389/fgeed.2021.617553.91ad17440a604c1d8fdfcd1d577555ac [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Tang X, Sretenovic S, Ren Q, et al. Plant prime editors enable precise gene editing in rice cells. Mol Plant. 2020;13:667–670. doi: 10.1016/j.molp.2020.03.010. [DOI] [PubMed] [Google Scholar]
  • 10.Hua K, Jiang Y, Tao X, Zhu JK. Precision genome engineering in rice using prime editing system. Plant Biotechnol J. 2020;18:2167–2169. doi: 10.1111/pbi.13395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Xu R, Li J, Liu X, Shan T, Qin R, Wei P. Development of plant prime-editing systems for precise genome editing. Plant Commun. 2020;1:100043. doi: 10.1016/j.xplc.2020.100043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Jiang YY, Chai YP, Lu MH, et al. Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize. Genome Biol. 2020;21:257. doi: 10.1186/s13059-020-02170-5.5ae751de190c46b0a5d55075e84a8549 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Butt H, Rao GS, Sedeek K, Aman R, Kamel R, Mahfouz M. Engineering herbicide resistance via prime editing in rice. Plant Biotechnol J. 2020;18:2370–2372. doi: 10.1111/pbi.13399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Yoo SD, Cho YH, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc. 2007;2:1565–1572. doi: 10.1038/nprot.2007.199. [DOI] [PubMed] [Google Scholar]
  • 15.Lee JS, Bae SJ, Kim JS, Kim C, Kang BC. A streamlined guide RNA screening system for genome editing in Sorghum bicolor. Plant Methods. 2023;19:90. doi: 10.1186/s13007-023-01058-2.2d4e2304a88f4c6d87518d0d780a9c87 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Jeong YY, Lee HY, Kim SW, Noh YS, Seo PJ. Optimization of protoplast regeneration in the model plant Arabidopsis thaliana. Plant Methods. 2021;17:21. doi: 10.1186/s13007-021-00720-x.8c8f9969870e470fba35b488d22a9756 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Xu W, Yang Y, Yang B, et al. A design optimized prime editor with expanded scope and capability in plants. Nat Plants. 2022;8:45–52. doi: 10.1038/s41477-021-01043-4. [DOI] [PubMed] [Google Scholar]
  • 18.Zhang X, Henriques R, Lin SS, Niu QW, Chua NH. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc. 2006;1:641–646. doi: 10.1038/nprot.2006.97. [DOI] [PubMed] [Google Scholar]
  • 19.Qiao D, Wang J, Lu MH, et al. Optimized prime editing efficiently generates heritable mutations in maize. J Integr Plant Biol. 2023;65:900–906. doi: 10.1111/jipb.13428. [DOI] [PubMed] [Google Scholar]
  • 20.Liang Z, Wu Y, Guo Y, Wei S. Addition of the T5 exonuclease increases the prime editing efficiency in plants. J Genet Genomics. 2023;50:582–588. doi: 10.1016/j.jgg.2023.03.008. [DOI] [PubMed] [Google Scholar]
  • 21.Ni P, Zhao Y, Zhou X, et al. Efficient and versatile multiplex prime editing in hexaploid wheat. Genome Biol. 2023;24:156. doi: 10.1186/s13059-023-02990-1.4682f652a27d469080bf348218d7cd35 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Chen R, Cao Y, Liu Y, et al. Enhancement of a prime editing system via optimal recruitment of the pioneer transcription factor P65. Nat Commun. 2023;14:257. doi: 10.1038/s41467-023-35919-0.a9653fd8e62547feadfa9b23d852884b [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ivanov S, Harrison MJ. A set of fluorescent protein-based markers expressed from constitutive and arbuscular mycorrhiza-inducible promoters to label organelles, membranes and cytoskeletal elements in Medicago truncatula. Plant J. 2014;80:1151–1163. doi: 10.1111/tpj.12706. [DOI] [PubMed] [Google Scholar]
  • 24.Yun LJ, Chen WL. SA and ROS are involved in methyl salicylate-induced programmed cell death in Arabidopsis thaliana. Plant Cell Rep. 2011;30:1231–1239. doi: 10.1007/s00299-011-1031-0. [DOI] [PubMed] [Google Scholar]
  • 25.Hwang GH, Jeong YK, Habib O, et al. PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing. Nucleic Acids Res. 2021;49:W499–W504. doi: 10.1093/nar/gkab319. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from BMB Reports are provided here courtesy of Korean Society for Biochemistry and Molecular Biology

RESOURCES