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Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.
Plant Cell Reports ( IF 6.2 ) Pub Date : 2019-11-14 , DOI: 10.1007/s00299-019-02488-w
Wusheng Liu 1, 2 , Mary R Rudis 1 , Matthew H Cheplick 1 , Reginald J Millwood 1 , Jian-Ping Yang 3 , Christine A Ondzighi-Assoume 1, 4 , Garrett A Montgomery 1 , Kellie P Burris 1, 5 , Mitra Mazarei 1 , Jonathan D Chesnut 3 , Charles Neal Stewart 1, 6
Affiliation  

A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 'Bright Yellow-2' (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

中文翻译:

脂质转染介导的基因组编辑,使用将 Cas9/gRNA 核糖核蛋白无 DNA 递送到植物细胞中。

一种新的、稳健的脂转染介导的转染方法,用于使用无 DNA 的 Cas9/gRNA RNP 进行基因编辑,已在植物细胞中证明了疗效。CRISPR/Cas9 系统彻底改变了精确的基因组编辑。基于 DNA 的 CRISPR/Cas9 递送被广泛用于各种植物物种。然而,基于蛋白质的体外翻译 Cas9/引导 RNA (gRNA) 核糖核蛋白 (RNP) 复合物向植物细胞的递送仍处于起步阶段,尽管蛋白质递送具有几个优势。这些优势包括无 DNA 递送、非转基因的基因编辑宿主植物、易用性、低成本、相对容易适应高通量系统以及低脱靶切割率。这里,我们展示了一种新的脂质转染介导的转染方法,用于将预组装的 Cas9/gRNA RNP 蛋白质递送到植物细胞中进行基因组编辑。两种脂质转染试剂,Lipofectamine 3000 和 RNAiMAX,适用于成功地将 Cas9/gRNA RNP 递送到植物细胞中。将绿色荧光蛋白 (GFP) 报告基因与 Cas9 蛋白的 C 端框内融合,并将融合蛋白成功递送到非转基因烟草品种中。'亮黄色-2' (BY2) 原生质体。Lipofectamine 3000 和 RNAiMAX 介导的蛋白质递送的最佳效率分别为 66% 和 48%。此外,我们基于已知的蛋白质组学技术开发了一种用于蛋白质递送的基因组学方法。表达橙色荧光蛋白报告基因 pporRFP 的转基因烟草 BY2 系被靶向用于敲除。我们发现 Lipofectamine 3000 介导的蛋白质递送的靶向诱变频率为 6%。我们的研究结果表明,新开发的脂转染介导的转染方法对于使用无 DNA 的 Cas9/gRNA 技术在植物细胞中进行基因组编辑是稳健的。
更新日期:2019-11-14
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