香蕉原生质体流线型转染系统体内验证及无转基因基因组编辑。

IF 2 3区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Hiralben Lakhani, Naveen Kumar, Alka Jangra, Sanjana Negi, Thobhanbhai Dholariya, Siddharth Tiwari
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引用次数: 0

摘要

CRISPR/Cas系统的进步极大地简化了植物基因组编辑,使其变得简单、可靠和高效。然而,对于香蕉这样的无性繁殖植物来说,开发无转基因作物是一项具有挑战性的任务。在本研究中,我们建立了基于香蕉原生质体的多功能高效基因组编辑平台来克服这一限制。本研究以香蕉品种大奈为研究对象,以叶片和胚性细胞悬浮(ECS)为研究对象,优化了原生质体分离方案。新鲜制备的ECS被认为是原生质体分离的最佳来源。通过转染质粒和RNP复合物检测原生质体的活力和能力。pCAMBIA1302和pJL50TRBO载体转染聚乙二醇(PEG)介导的原生质体,分别以30%和70%的效率表达GFP,最终证明了该方案的有效性。此外,通过体外裂解实验验证了靶向香蕉β-胡萝卜素羟化酶基因的gRNAs,并将其用于SpCas9与gRNA1的不同比例(1:1,1:2,1:5和1:10)的RNP复合物形成。其中,当摩尔比为1:2时,产生的indel频率为7%。目标扩增子的测序分析显示,在三种体外验证的grna中,PAM区域上游发生突变,特别是gRNA1。本研究评估了grna在体外和体内的有效性,结果不一致,强调需要对其功能进行全面的体内验证。总之,香蕉转染的优化方案有潜力被利用来产生无转基因的转基因改良香蕉。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Streamlined protoplast transfection system for in-vivo validation and transgene-free genome editing in Banana.

The advancement in the CRISPR/Cas system has significantly streamlined genome editing in plants, rendering it simple, reliable, and efficient. However, the development of transgene-free crops is a challenging task for vegetatively propagated plants like banana. In the present study, we established banana protoplasts-based versatile and efficient platform for genome editing to overcome this limitation. Herein, a protocol has been optimized for protoplast isolation by considering leaf and embryogenic cell suspension (ECS) of banana cultivar Grand Naine. Freshly prepared ECS was identified as the best source for protoplast isolation. The protoplast viability and competency were checked by transfection with plasmid and RNP complex. Polyethylene glycol (PEG)-mediated protoplast transfection using pCAMBIA1302 and pJL50TRBO vectors showed GFP expression with 30 and 70% efficiency, respectively, eventually proving the protocol's efficacy. Further, gRNAs targeting banana β-carotene hydroxylase gene are validated by in-vitro cleavage test and subsequently used for RNP complex formation with varied ratios (1:1, 1:2, 1:5, and 1:10) of SpCas9 to gRNA1. Among these, a 1:2 molar ratio proved best to generate indel frequency with 7%. Sequencing analysis of the target amplicon revealed mutations upstream of the PAM region, specifically with gRNA1, among the three in-vitro validated gRNAs. This study evaluated the effectiveness of gRNAs in-vitro and in-vivo, yielding inconsistent results that highlight the need for comprehensive in-vivo validation of their functionality. Conclusively, the optimized protocol for banana transfection has the potential to be harnessed for the generation of transgene-free genetically improved banana.

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来源期刊
Transgenic Research
Transgenic Research 生物-生化研究方法
CiteScore
5.40
自引率
0.00%
发文量
38
审稿时长
4-8 weeks
期刊介绍: Transgenic Research focusses on transgenic and genome edited higher organisms. Manuscripts emphasizing biotechnological applications are strongly encouraged. Intellectual property, ethical issues, societal impact and regulatory aspects also fall within the scope of the journal. Transgenic Research aims to bridge the gap between fundamental and applied science in molecular biology and biotechnology for the plant and animal academic and associated industry communities. Transgenic Research publishes -Original Papers -Reviews: Should critically summarize the current state-of-the-art of the subject in a dispassionate way. Authors are requested to contact a Board Member before submission. Reviews should not be descriptive; rather they should present the most up-to-date information on the subject in a dispassionate and critical way. Perspective Reviews which can address new or controversial aspects are encouraged. -Brief Communications: Should report significant developments in methodology and experimental transgenic higher organisms
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