Lin Wu, Jun Yang, Yuying Gu, Qianyi Wang, Zeyu Zhang, Hongjue Guo, Liangzhen Zhao, Hangxiao Zhang, Lianfeng Gu
{"title":"Bamboo mosaic virus-mediated transgene-free genome editing in bamboo","authors":"Lin Wu, Jun Yang, Yuying Gu, Qianyi Wang, Zeyu Zhang, Hongjue Guo, Liangzhen Zhao, Hangxiao Zhang, Lianfeng Gu","doi":"10.1111/nph.20386","DOIUrl":null,"url":null,"abstract":"<p>The common method of delivering CRISPR/Cas reagents for genome editing in plants involves <i>Agrobacterium</i>-mediated transformation or preassembled CRISPR/Cas9 ribonucleoprotein complex delivery (Woo <i>et al</i>., <span>2015</span>; Toda <i>et al</i>., <span>2019</span>; Ye <i>et al</i>., <span>2020</span>). These methods require labor-intensive and time-consuming plant tissue culture processes (Huang <i>et al</i>., <span>2022</span>). Unfortunately, most plants exhibit extremely low efficiency in callus induction and regeneration; these technical challenges greatly hinder the application of genome editing. Recent developments in plant RNA virus-based expression vectors (Ma <i>et al</i>., <span>2020</span>; Chen <i>et al</i>., <span>2022</span>) provide a convenient, efficient, and cost-effective way for DNA-free genome editing in plants, leveraging the fact that virus RNA does not integrate into the genome. However, the stability of virus vectors is negatively correlated with the size of the inserted foreign genes. Consequently, achieving efficient expression of <i>Streptococcus pyogenes</i> Cas9 (<i>SpCas9</i>, <i>c</i>. 4.2 kb) by virus-based vectors remains challenging. Most reported viruses capable of delivering Cas9 proteins are negative-strand RNA viruses (Ma <i>et al</i>., <span>2020</span>; Liu <i>et al</i>., <span>2023</span>; Zhao <i>et al</i>., <span>2024</span>), with only a few positive-strand RNA viruses identified (Uranga <i>et al</i>., <span>2021</span>; Lee <i>et al</i>., <span>2024</span>). Thus, delivering virus-based sgRNA vectors to plants overexpressing Cas9 is the most commonly used strategy (Ali <i>et al</i>., <span>2015</span>; Jiang <i>et al</i>., <span>2019</span>; Li <i>et al</i>., <span>2021</span>). However, it is difficult to use this method to generate a Cas9-free mutant by crossing with wild-type (WT) plants with long flowering cycles, such as bamboo (Ye <i>et al</i>., <span>2017</span>). <i>Bamboo mosaic virus</i> (BaMV) has a typical flexible filamentous virion structure with the positive-sense single-stranded RNA genome (Hsu <i>et al</i>., <span>2018</span>). The BaMV-mediated expression system can effectively drive the expression of large foreign gene fragments (Jin <i>et al</i>., <span>2023</span>). For the first time, we developed a BAMV-mediated Cas protein and sgRNA delivery system in WT <i>Nicotiana benthamiana</i> and bamboo. This approach enables targeted gene editing in noninfected leaves or stems in bamboo without the need for Cas9-expressing transgenic lines, leveraging BaMV's large cargo ability to transport Cas9 proteins.</p><p>None declared.</p><p>LG conceived and designed the research. LW, JY, YG, QW, HG and LZ performed experiments. ZZ and HZ performed the bioinformatics analyses. LW and LG prepared the manuscript. LW, JY and YG contributed equally to this work.</p><p>The New Phytologist Foundation remains neutral with regard to jurisdictional claims in maps and in any institutional affiliations.</p>","PeriodicalId":214,"journal":{"name":"New Phytologist","volume":"245 5","pages":"1810-1816"},"PeriodicalIF":8.3000,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/nph.20386","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"New Phytologist","FirstCategoryId":"99","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/nph.20386","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
引用次数: 0
Abstract
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 et al., 2019; Ye et al., 2020). These methods require labor-intensive and time-consuming plant tissue culture processes (Huang et al., 2022). Unfortunately, most plants exhibit extremely low efficiency in callus induction and regeneration; these technical challenges greatly hinder the application of genome editing. Recent developments in plant RNA virus-based expression vectors (Ma et al., 2020; Chen et al., 2022) provide a convenient, efficient, and cost-effective way for DNA-free genome editing in plants, leveraging the fact that virus RNA does not integrate into the genome. However, the stability of virus vectors is negatively correlated with the size of the inserted foreign genes. Consequently, achieving efficient expression of Streptococcus pyogenes Cas9 (SpCas9, c. 4.2 kb) by virus-based vectors remains challenging. Most reported viruses capable of delivering Cas9 proteins are negative-strand RNA viruses (Ma et al., 2020; Liu et al., 2023; Zhao et al., 2024), with only a few positive-strand RNA viruses identified (Uranga et al., 2021; Lee et al., 2024). Thus, delivering virus-based sgRNA vectors to plants overexpressing Cas9 is the most commonly used strategy (Ali et al., 2015; Jiang et al., 2019; Li et al., 2021). However, it is difficult to use this method to generate a Cas9-free mutant by crossing with wild-type (WT) plants with long flowering cycles, such as bamboo (Ye et al., 2017). Bamboo mosaic virus (BaMV) has a typical flexible filamentous virion structure with the positive-sense single-stranded RNA genome (Hsu et al., 2018). The BaMV-mediated expression system can effectively drive the expression of large foreign gene fragments (Jin et al., 2023). For the first time, we developed a BAMV-mediated Cas protein and sgRNA delivery system in WT Nicotiana benthamiana and bamboo. This approach enables targeted gene editing in noninfected leaves or stems in bamboo without the need for Cas9-expressing transgenic lines, leveraging BaMV's large cargo ability to transport Cas9 proteins.
None declared.
LG conceived and designed the research. LW, JY, YG, QW, HG and LZ performed experiments. ZZ and HZ performed the bioinformatics analyses. LW and LG prepared the manuscript. LW, JY and YG contributed equally to this work.
The New Phytologist Foundation remains neutral with regard to jurisdictional claims in maps and in any institutional affiliations.
用于植物基因组编辑的CRISPR/Cas试剂的常见递送方法包括农杆菌介导的转化或预组装的CRISPR/Cas9核糖核蛋白复合物递送(Woo et al., 2015;Toda等人,2019;Ye et al., 2020)。这些方法需要劳动密集型和耗时的植物组织培养过程(Huang et al., 2022)。然而,大多数植物的愈伤组织诱导和再生效率极低;这些技术挑战极大地阻碍了基因组编辑的应用。植物RNA病毒表达载体的最新进展(Ma et al., 2020;Chen et al., 2022)利用病毒RNA不整合到基因组的事实,为植物的无dna基因组编辑提供了一种方便、高效、经济的方法。然而,病毒载体的稳定性与插入的外源基因的大小呈负相关。因此,通过基于病毒的载体实现化脓性链球菌Cas9 (SpCas9,约4.2 kb)的高效表达仍然具有挑战性。大多数报道的能够传递Cas9蛋白的病毒是负链RNA病毒(Ma et al., 2020;Liu et al., 2023;Zhao et al., 2024),仅鉴定出少数正链RNA病毒(Uranga et al., 2021;Lee et al., 2024)。因此,将基于病毒的sgRNA载体传递给过表达Cas9的植物是最常用的策略(Ali et al., 2015;Jiang et al., 2019;Li等人,2021)。然而,这种方法很难通过与长开花周期的野生型(WT)植物(如竹子)杂交来产生无cas9突变体(Ye et al., 2017)。竹花叶病毒(BaMV)具有典型的柔性丝状病毒粒子结构,具有正义单链RNA基因组(Hsu et al., 2018)。bamv介导的表达系统可以有效地驱动外源大片段基因的表达(Jin et al., 2023)。本研究首次在野生型烟叶和竹子中建立了bamv介导的Cas蛋白和sgRNA传递系统。这种方法能够在未感染的竹子叶片或茎中进行靶向基因编辑,而不需要表达Cas9的转基因品系,利用BaMV运输Cas9蛋白的大货物能力。
期刊介绍:
New Phytologist is an international electronic journal published 24 times a year. It is owned by the New Phytologist Foundation, a non-profit-making charitable organization dedicated to promoting plant science. The journal publishes excellent, novel, rigorous, and timely research and scholarship in plant science and its applications. The articles cover topics in five sections: Physiology & Development, Environment, Interaction, Evolution, and Transformative Plant Biotechnology. These sections encompass intracellular processes, global environmental change, and encourage cross-disciplinary approaches. The journal recognizes the use of techniques from molecular and cell biology, functional genomics, modeling, and system-based approaches in plant science. Abstracting and Indexing Information for New Phytologist includes Academic Search, AgBiotech News & Information, Agroforestry Abstracts, Biochemistry & Biophysics Citation Index, Botanical Pesticides, CAB Abstracts®, Environment Index, Global Health, and Plant Breeding Abstracts, and others.