Bamboo mosaic virus‐mediated transgene‐free genome editing in bamboo

New Phytologist, Journal Year: 2025, Volume and Issue: 245(5), P. 1810 - 1816

Published: Jan. 6, 2025

The common method of delivering CRISPR/Cas reagents for genome editing in plants involves Agrobacterium-mediated transformation or preassembled CRISPR/Cas9 ribonucleoprotein complex delivery (Woo et al., 2015; Toda 2019; Ye 2020). These methods require labor-intensive and time-consuming plant tissue culture processes (Huang 2022). Unfortunately, most exhibit extremely low efficiency callus induction regeneration; these technical challenges greatly hinder the application editing. Recent developments RNA virus-based expression vectors (Ma 2020; Chen 2022) provide a convenient, efficient, cost-effective way DNA-free plants, leveraging fact that virus does not integrate into genome. However, stability is negatively correlated with size inserted foreign genes. Consequently, achieving efficient Streptococcus pyogenes Cas9 (SpCas9, c. 4.2 kb) by remains challenging. Most reported viruses capable proteins are negative-strand Liu 2023; Zhao 2024), only few positive-strand identified (Uranga 2021; Lee 2024). Thus, sgRNA to overexpressing commonly used strategy (Ali Jiang Li 2021). it difficult use this generate Cas9-free mutant crossing wild-type (WT) long flowering cycles, such as bamboo (Ye 2017). Bamboo mosaic (BaMV) has typical flexible filamentous virion structure positive-sense single-stranded (Hsu 2018). BaMV-mediated system can effectively drive large gene fragments (Jin 2023). For first time, we developed BAMV-mediated Cas protein WT Nicotiana benthamiana bamboo. This approach enables targeted noninfected leaves stems without need Cas9-expressing transgenic lines, BaMV's cargo ability transport proteins. seeds were germinated on 1/2 MS medium (M519; Phytotech, Lenexa, KS, USA), supplemented 30 g l−1 sucrose, cultured at 28°C growth chamber 16 h : 8 h, light dark photoperiod. green seedlings then transferred soil cultivated glasshouse maintained 23–25°C 60% relative humidity 15 9 Dendrocalamus latiflorus Munro Phyllostachys edulis, peeled soaked water 24 before being evenly sown uniformly nutrient-enriched covered 1-cm thick nutrient soil. under To construct BaMV expressing Cas9, two variants AsCas12f1 sgRNA, modified previously BaMV-Cas9 vector involved addition coat (CP) promoter an Xba I restriction site following termination codon triple block 3 (TGBp3). We 83 bp scaffold (for Cas9) 94 HKRA YHAM) guide sequences specific N. (gNbPDS), P. edulis (gPheRDR6) D. (gDlmRDR6) (Supporting Information Table S1; Fig. S1a) site, control CP promoter. constructed vector, containing SpCas9 was designated pBaMV-Cas9, sgRNA. pBaMV-HKRA pBaMV-YHAM (Figs 1a, S2). Previous studies have shown synthetic constructs tandemly arrayed tRNA-gRNA structures precise processing gRNAs achieve simultaneous targeting multiple genetic loci (Xie 2015). Additionally, also designed pBaMV-g1tg2t-Cas9 multiplexed attaching 77-bp tRNAGly sequence (Ellison 2020) 3′ end concatenated sgRNAs sgRNA1-tRNAGly-sgRNA2-tRNAGly (g1tg2t) (Fig. 1a; S1). pBaMV-nCas9 pBaMV-cCas9 vectors, split 573-aa nCas9 794-aa cCas9 (Truong 2015), fused Npu_DnaE (nIntein cIntein) split-Cas9. Split-Cas9 after fusion separately assembled subgenome pBaMV-Cas9 pBaMV-AsCas12f1 plasmids Agrobacterium tumefaciens strain GV3101 using freeze–thaw method. plasmid Yeast Extract Mannitol Broth (YEB) solid 50 mg kanamycin 25 rifampicin 48 h. A single colony selected inoculated liquid same concentration antibiotics. shaken incubated 200 rpm until OD600 0.5–0.8 reached. Enriching bacteria centrifuged briefly washed once tobacco infection buffer (10 mM MES, 10 MgCl2, 100 μM acetylsyringane, pH 5.6). Finally, resuspended 25°C darkness 2 inoculating leaves. After d infiltration, infected ground (1% PVP, tripotassium orthophosphate, 1‰ 2-Hydroxy-1-ethanethiol, 8.0) diamond sand. Then, juice obtained from grinding mechanical inoculation moisturizing cultivation, returned normal conditions 23–25°C. Total extracted TRIZOL 0.1 fresh leaf stem spectrophotometer (DS-11 FX+; DeNovix, Wilmington, DE, USA) quantitative assessment. According protocol HiScript II 1st Strand cDNA Synthesis Kit (R211-01; Vazyme, Nanjing, China), first-stranded synthesized μg total treated DNase, oligo-dT primers 3′-UTR (Ba32). All primer reverse transcription polymerase chain reaction (RT-PCR) analysis listed S1. provided, DNA extraction performed 0.15 HiPure HP Plant (D3187; Magen, Beijing, China). PCR amplification target genes samples, high-fidelity enzyme × Phanta Flash Master Mix (Dye Plus) (P520; Vazyme) used. purification, products subjected restrictive digestion Nco (FD0574; Thermo Scientific, Colorado Springs, CO, T7 Endonuclease (EN303; Vazyme). resulting digested detected via electrophoresis 2% agarose gel. Sanger sequencing, ng corresponding enzymes, followed TOPO-cloning easy Universal Zero TOPO TA/Blunt Cloning (10906ES20; Yeasen, Shanghai, Six positive clones sequenced. amplicon deep Hi-TOM sequencing according Rapid Sequencing Service Protocol (http://www.hi-tom.net/hi-tom/) (Sun cloned sequenced, clean aligned reference Hi-Tom online platform. efficiencies calculated dividing number reads indels sequenced reads. extraction, samples BaMV-infected quickly frozen nitrogen powder. Subsequently, 300 μl loading (25 Tris–HCl 6.8, 0.5 M DTT, 10% (w/v) SDS, 0.5% bromophenol blue, 50% (v/v) glycerol) added each sample, mixture homogenized heated 100°C min. room temperature 12 396 Proteins separated 12.5% SDS-polyacrylamide gel subsequently onto nitrocellulose (NC) membrane V 90 min 1× transfer Tris base, 192 glycine, 3.5 20% solvent). Following transfer, NC salt solution (TBS, 20 150 NaCl, 7.5) once, seal TBST (TBS 0.05% Tween 20) 5% skim milk powder 1 gentle agitation rpm. closed TBS primary antibody temperature. three washes each), secondary antibodies underwent additional final rinse TBS. chemiluminescent substrate analyzed Amersham Imager 600. Ordinary one-way ANOVA two-way tests GraphPad prism v.10.2.0 (GraphPad Software Inc., San Diego, CA, comparative across different groups. address limitations low-genetic during regeneration, engineered integrating between TGBps 1a). Heterologous controlled further enhance infectivity increase levels incorporated p19 Tombusvirus, linked 2A peptide. assess feasibility BaMV-based mutagenesis endogenous benthamiana, Phytoene Desaturase (PDS) (Table S1a). Four-week-old infiltrated carrying NbPDS 1b). Approximately wk examined fifth sixth symptomatic systemic (showing curling chlorosis) molecular level measure viral RNA, transcripts, 1c,d). PCR-RE assays utilizing endonuclease revealed frequencies ranging 19% 37% NbPDS-A 29% 52% NbPDS-B 1e, S1b). truncated showed mediated ranged 35.8% 47.4%, average 41.3% 1f). confirmed various types 1g, S1c; Notes postinoculation (dpi) detection leaves, which consistent mutations well S1d). evaluate multiplex system, either distinct sites within (gNbPDS&gNbPDS-2) both PDS RNA-dependent Polymerase 6 (RNR6) (gNbPDS&gNbRDR6) Two identical 77-base pair (bp) appended form pBaMV-g1tg2t-Cas9, collected detection, results could induce effective despite reduced compared pBaMV-Cas9. gNbPDS&gNbPDS-2 vector-mediated mutation 12% 15% 14% 23%, respectively 1h, S1e). Amplicon 4.14% 5.79% (average 4.93%), similarly, 5.49% 8.72% 7.41%) 1i). successfully caused changes NbPDS-B, along fragment deletions cleavage 1j; based NbRDR6-B gNbPDS&gNbRDR6 21% 34% 26%, 2a, S1f). 7.39% 16.88% 12.27%), while NbRDR6, 12.09% 16.67% 14.26%) 2b). Simultaneously, NbRDR6 2c, S1g; substantiate efficacy DlmRDR6 PheRDR6 loci, respectively. dpi, newly grown exhibited symptoms 1b), immunoblotting new S2a). transcripts assessed RT-PCR S2b). (T7EI) indicated successful In latiflorus, 9% 6% 16% 2d, S2c). Similarly, 4% 2e, S2d). validate T7EI results, conducted one 520-bp product 538-bp PheRDR6. 2.01% 5.81% 3.34%) 2.52% 7.56% 4.15%) 2f). PheRDR6, 1.82% 8.88% 4.89%) 1.28% 7.78% 4.28%) 2g). Examination near insertions (Indels), confirming 2h,i). dpi S2e,f), well. explored co-expressed systems expressed isolated (complete version split-Cas9) S2g). found co-expression be injected split-Cas9 did yield detectable S2h). AsCas12f1, namely Ascas12f1-YHAM Ascas12f1-HKRA, higher rice NbPDS. AsCas12f1-HKRA had than AsCas12f1-YHAM 2j). 17.42% 31.14% 26.27%), 1.19% 1.96% 1.72%). Although significantly lower 2k), able 2l, S2i; S1), suggesting significant potential deletion AsCas12f1-HKRA. summary, achieved demonstrating research. current shows relatively our study broadens library gene-editing tools positive-stranded holds advance VIGE technology plants. aim improve focusing optimization exploring other future studies. Furthermore, due spread widely BaMV-induced may facilitate more rapid acquisition resources through regeneration. Notably, broad host range, encompasses monopodial sympodial bamboo, expanding prospects breeding improvement. research funded National Key R&D Program China (2021YFD2200505), Natural Science Foundation (3237141305), Fujian Province (2021J02027), S&T Innovation (KFB23180) Forestry Peak Discipline Construction Project Agriculture University (72202200205). None declared. LG conceived LW, JY, YG, QW, HG LZ experiments. ZZ HZ bioinformatics analyses. LW prepared manuscript. JY YG contributed equally work. data supports findings available Supporting article. Supplementary figures related Figs S1 S2. information required experimental integrity, refer efficiency, including showing frequencies. S2 edulis. Results sequencing. Oligos, relevant study. Please note: Wiley responsible content functionality any supplied authors. Any queries (other missing material) should directed New Phytologist Central Office. publisher supporting content) author neutral regard jurisdictional claims maps institutional affiliations.

Language: Английский

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Current Advancement and Future Prospects in Simplified Transformation-Based Plant Genome Editing

Plants, Journal Year: 2025, Volume and Issue: 14(6), P. 889 - 889

Published: March 12, 2025

Recent years have witnessed remarkable progress in plant biology, driven largely by the rapid evolution of CRISPR/Cas-based genome editing (GE) technologies. These tools, including versatile CRISPR/Cas systems and their derivatives, such as base editors prime editors, significantly enhanced universality, efficiency, convenience functional genomics, genetics, molecular breeding. However, traditional genetic transformation methods are essential for obtaining GE plants. depend on tissue culture procedures, which time-consuming, labor-intensive, genotype-dependent, challenging to regenerate. Here, we systematically outline current advancements simplifying GE, focusing optimization process through developmental regulators, development planta methods, establishment nanomaterial- viral vector-based delivery platforms. We also discuss critical challenges future directions achieving genotype-independent, culture-free GE.

Language: Английский

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An integrative overview of cold response and regulatory pathways in horticultural crops

Journal of Integrative Plant Biology, Journal Year: 2025, Volume and Issue: unknown

Published: April 11, 2025

ABSTRACT Global climate change challenges agricultural production, as extreme temperature fluctuations negatively affect crop growth and yield. Low (LT) stress impedes photosynthesis, disrupts metabolic processes, compromises the integrity of cell membranes, ultimately resulting in diminished yield quality. Notably, many tropical or subtropical horticultural plants are particularly susceptible to LT stress. To address these challenges, it is imperative understand mechanisms underlying cold tolerance crops. This review summarizes recent advances physiological molecular that enable crops withstand stress, emphasizing discrepancies between model systems. These include C‐repeat binding factor‐dependent transcriptional regulation, post‐translational modifications, epigenetic control, regulation. Reactive oxygen species, plant hormones, light signaling pathways integrated into response network. Furthermore, technical for improving highlighted, including genetic improvement, application light‐emitting diodes, utility novel regulators, grafting. Finally, prospective directions fundamental research practical applications boost discussed.

Language: Английский

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Establishment of a Genetic Transformation and Gene Editing Method by Floral Dipping in Descurainia sophia

Plants, Journal Year: 2024, Volume and Issue: 13(20), P. 2833 - 2833

Published: Oct. 10, 2024

L. Webb ex Prantl is used in traditional medicine globally. However, the lack of an efficient and reliable genetic transformation system has seriously limited investigation gene function further utilization

Language: Английский

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Protocol for transformation-free genome editing in plants using RNA virus vectors for CRISPR-Cas delivery

STAR Protocols, Journal Year: 2024, Volume and Issue: 5(4), P. 103437 - 103437

Published: Nov. 5, 2024

Language: Английский

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Development of an RNA virus vector for non-transgenic genome editing in tobacco and generation of berberine bridge enzyme-like mutants with reduced nicotine content

aBIOTECH, Journal Year: 2024, Volume and Issue: 5(4), P. 449 - 464

Published: Nov. 22, 2024

Tobacco (

Language: Английский

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