Nanomaterials Enable Delivery of Genetic Material Without Transgene Integration in Mature Plants
纳米材料应用于非转基因成熟植物中遗传物质的传递
Markita Landry
ABSRACT
Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis, and agricultural crop engineering. The plant cell wall is a barrier that limits the ease and throughput with which exogenous biomolecules can be delivered to plants. Current delivery methods either suffer from host range limitations, low transformation efficiencies, tissue regenerability, tissue damage, or unavoidable DNA integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into tissues and organs of intact plants of several species with a suite of pristine and chemically-functionalized high aspect ratio nanomaterials [1]. Efficient DNA delivery and strong protein expression without transgene integration is accomplished in mature Nicotiana benthamiana, Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts [2]. Notably, we demonstrate that transgene expression is transient and devoid of transgene integration into the plant host genome, of potential utility for easing regulatory oversight of transformed crops as genetically modified organisms (GMOs) [3]. We demonstrate that our platform can be applied for CRISPR-based genome editing for transient expression of Cas9 and gRNAs. We also demonstrate a second nanoparticle-based strategy in which small interfering RNA (siRNA) is delivered to mature Nicotiana benthamiana leaves and effectively silence a gene with 95% efficiency. We find that nanomaterials both facilitate biomolecule transport into plant cells, while also protecting polynucleotides such as RNA from nuclease degradation. DNA origami and nanostructures further enable siRNA delivery to plants at programmable nanostructure loci [4], which we use to elucidate force-independent transport phenomena of nanoparticles across the plant cell wall [5]. Our work provides a tool for species-independent, targeted, and passive delivery of genetic material, without transgene integration, into plant cells for diverse plant biotechnology applications.
植物基因工程是可持续发展、天然产物合成和农作物工程的核心技术。植物细胞壁是限制外源性生物分子传输的屏障。目前的传递方法要么受到宿主范围、转化效率低、组织不可再生、组织损伤等的限制，要么将不能废除的DNA整合到宿主基因组中。在本次讲座中，我们将展示利用系列新型化学修饰的高深宽比的纳米材料，以扩散为基础，将生物分子原封不动地传递到植物的组织和器官中。在成熟的烟草、芥菜、小麦和棉花的叶片和芝麻原生质体中实现了没有转基因整合的高效的DNA传递和高蛋白表达。这里值得注意的是，我们的结果表明这种基因表达是瞬时的，并且没有转基因整合到植物的宿主基因组中，这对于减轻转基因作物作为转基因生物（GMOs）的监管具有潜在的实用价值。我们的平台可用于基于CRISPR的基因组编辑对Cas9和gRNA进行瞬时表达。我们还演示了第二种基于纳米粒子的策略，其中将小干扰RNA（siRNA）传递至成熟的本生烟草叶，并能以95％的效率有效沉默一种基因。我们发现纳米材料既能促进生物分子向植物细胞内的运输，又能保护诸如RNA的多核苷酸免受核酸酶降解。DNA双螺旋结构和纳米结构能够促进siRNA在可编程纳米结构位点上转移到植物，运用这一点我们阐明了纳米粒子在植物细胞壁上不依赖于力的转运现象。综上所述，我们可在不进行转基因整合的情况下，将外源性生物分子不依赖于物种且定向和被动地传递给多种植物细胞，这为植物生物技术提供了一种实用工具。
BIOGRAPHY
Markita Landry is an assistant professor in the department of Chemical and Biomolecular Engineering at the University of California, Berkeley. She received a B.S. in Chemistry, and a B.A. in Physics from the University of North Carolina at Chapel Hill, a Ph.D. in Chemical Physics and a Certificate in Business Administration from the University of Illinois at Urbana-Champaign, and completed an NSF postdoctoral fellowship in Chemical Engineering at the Massachusetts Institute of Technology. Her current research centers on the development of synthetic nanoparticle-polymer conjugates for imaging neuromodulation in the brain, and for the delivery of genetic materials into plants for plant biotechnology applications. The Landry lab exploits the highly tunable chemical and physical properties of nanomaterials for the creation of bio-mimetic structures, molecular imaging, and plant genome editing. She is also on the scientific advisory board of Terramera, Inc, and on the scientific advisory board of Chi-Botanic. She is a recent recipient of 20 early career awards, including awards from the Brain and Behavior Research Foundation, the Burroughs Wellcome Fund, The Parkinson’s Disease Foundation, the DARPA Young Investigator program, the Beckman Young Investigator program, the Howard Hughes Medical Institute, is a Sloan Research Fellow, an FFAR New Innovator, and is a Chan-Zuckerberg Biohub Investigator.
Markita Landry是加利福尼亚大学伯克利分校化学与生物分子工程系的助理教授。她在北卡罗来纳大学教堂山分校获得了化学学士学位和文学学士学位，在伊利诺伊大学厄本那-香槟分校化学获得物理学博士学位和工商管理证书，并在麻省理工学院化学工程系完成NSF博士后研究。她目前的研究方向主要是开发用于成像的纳米粒子-聚合物共轭物在大脑中的神经调节，以及将遗传物质输送到植物中以进行植物生物技术应用。Landry实验室利用纳米材料的高度可调节的化学和物理特性来创建仿生结构、分子成像和植物基因组编辑。她担任Terramera公司和Chi-Botanic的科学顾问。近年来，她获得了20项早期职业奖项，其中包括来自大脑与行为研究基金会，伯勒斯惠康基金会，帕金森氏病基金会，DARPA青年研究人员计划，贝克曼青年研究人员计划，霍华德·休斯医学研究所等等的奖项。Landry还是Sloan研究会的资深会员，FFAR的新发明家，也是Chan-Zuckerberg Biohub的科学家。