Mahmoud Lamiaa M, Kaur Prabhjot, Stanton Daniel, Grosser Jude W, Dutt Manjul
Citrus Research and Education Center, University of Florida, Lake Alfred, FL, 33850, USA.
Pomology Department, Faculty of Agriculture, Mansoura University, Mansoura, Egypt.
Plant Methods. 2022 Mar 18;18(1):33. doi: 10.1186/s13007-022-00870-6.
The genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants.
Here, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1.
Our results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.
面对气候变化和全球人口增长，作物基因工程通过赋予理想的遗传性状，包括增强生物和非生物胁迫耐受性，提高了农业生产力。成簇规律间隔短回文重复序列（CRISPR/Cas9）系统已被发现是一种很有前景的基因组编辑技术。原生质体常被用于通过将重组DNA片段体外整合到植物基因组中来培育转基因植物。我们靶向柑橘病程相关蛋白3的非表达子（CsNPR3）基因，该基因是系统获得性抗性（SAR）的负调控因子，它控制蛋白酶体介导的NPR1降解，并开发了一种靶向柑橘原生质体DNA的基因组编辑技术，以产生稳定的基因组编辑柑橘植株。
在此，我们确定了用于递送供体DNA的最佳阳离子脂质纳米颗粒，并描述了一种使用Lipofectamine™ LTX试剂和PLUS试剂介导DNA导入柑橘原生质体的方案。使用阳离子脂质转染剂Lipofectamine，无论有无聚乙二醇（PEG，分子量6000），将含有靶向CsNPR3基因的gRNA的Cas9构建体转染到柑橘原生质体中。Lipofectamine中封装的最佳转染效率为30%，Lipofectamine与PEG组合时为51%，仅PEG时为2%。此外，与PEG相比，Lipofectamine中质粒封装导致的细胞活力百分比最高（45%）。通过T7EI分析和Sanger测序获得并鉴定了9株编辑植株。所培育的编辑株系表现出CsNPR3表达下调和CsPR1表达上调。
我们的结果表明，使用基于阳离子脂质的转染剂Lipofectamine是成功递送供体DNA并随后在柑橘中成功进行基因组编辑的可行选择。
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