An efficient mRNA delivery system for genome editing in plants

IF 10.1 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Fengti Qiu, Chenxiao Xue, Jinxing Liu, Boshu Li, Qiang Gao, Ronghong Liang, Kunling Chen, Caixia Gao
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Abstract

Transgene-free genome editing is important for crop improvement as it reduces unanticipated genomic changes. While mRNA delivery systems offer a powerful method for achieving transgene-free genome editing, they remain inefficient and challenging in plants. Here we describe an efficient mRNA delivery system for plants with substantially improved editing efficiency. By optimizing the 5′ untranslated regions (5′UTRs) and poly(A) tails of in vitro-transcribed (IVT) mRNAs and coating the mRNA with protamine during particle bombardment, we have developed an optimized mRNA delivery system termed v2_TMV/DEN2. This system enhanced the efficiencies of knock-out, A-to-G and C-to-T base editing by an average 4.7-, 3.4- and 2.5-fold at various endogenous sites compared with plasmid-based transient delivery system via particle bombardment in rice suspension cells and wheat immature embryos 48 h post-transformation. Furthermore, we obtained edited plants with efficiencies of 5.0–180.8% and 26.1–26.2% using v2_TMV/DEN2 in rice and wheat, respectively, compared with 0.0–43.2% and 4.7–10.4% using plasmids. Our study provides a convenient and efficient mRNA delivery system for transgene-free genome editing in plants.

Abstract Image

一种用于植物基因组编辑的高效mRNA传递系统
无转基因基因组编辑对作物改良很重要,因为它减少了意想不到的基因组变化。虽然mRNA传递系统为实现无转基因基因组编辑提供了一种强大的方法,但它们在植物中仍然效率低下且具有挑战性。在这里,我们描述了一种有效的植物mRNA传递系统,大大提高了编辑效率。通过优化体外转录(IVT) mRNA的5 ‘非翻译区(5 ’ utr)和多聚(A)尾部,并在粒子轰击期间用鱼精蛋白包裹mRNA,我们开发了一种优化的mRNA递送系统,称为v2_TMV/DEN2。与基于质粒的瞬时传递系统相比,该系统在水稻悬浮细胞和小麦未成熟胚胎转化48 h后,在不同内源位点上的敲除、A-to-G和C-to-T碱基编辑效率平均提高了4.7倍、3.4倍和2.5倍。此外,我们在水稻和小麦中使用v2_TMV/DEN2获得的编辑植株效率分别为5.0-180.8%和26.1-26.2%,而使用质粒获得的编辑植株效率分别为0.0-43.2%和4.7% - 10.4%。本研究为植物无转基因基因组编辑提供了一种便捷高效的mRNA传递系统。
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来源期刊
Plant Biotechnology Journal
Plant Biotechnology Journal 生物-生物工程与应用微生物
CiteScore
20.50
自引率
2.90%
发文量
201
审稿时长
1 months
期刊介绍: Plant Biotechnology Journal aspires to publish original research and insightful reviews of high impact, authored by prominent researchers in applied plant science. The journal places a special emphasis on molecular plant sciences and their practical applications through plant biotechnology. Our goal is to establish a platform for showcasing significant advances in the field, encompassing curiosity-driven studies with potential applications, strategic research in plant biotechnology, scientific analysis of crucial issues for the beneficial utilization of plant sciences, and assessments of the performance of plant biotechnology products in practical applications.
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