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RNA 沉默抑制子影响携带指导 RNA 和 Cas9 的病毒载体进行 CRISPR 基因编辑的性能。

RNA silencing suppressor-influenced performance of a virus vector delivering both guide RNA and Cas9 for CRISPR gene editing.

机构信息

Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX, 77843, USA.

Department of Surgery, Duke University School of Medicine, Durham, NC, 27710, USA.

出版信息

Sci Rep. 2021 Mar 24;11(1):6769. doi: 10.1038/s41598-021-85366-4.

Abstract

We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas9 components into plants. First, production of a Cas9 (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein. Such suppressor-generated elevated levels of Cas9 expression translated to efficient gene editing mediated by TRBO-G-3'gGFP expressing GFP and also a single guide RNA targeting the mgfp5 gene in the Nicotiana benthamiana GFP-expressing line 16c. Furthermore, HcoCas9 encoding RNA, a large cargo insert of 4.2 kb, was expressed from TRBO-HcoCas9 to yield Cas9 protein again at higher levels upon co-expression with P19. Likewise, co-delivery of TRBO-HcoCas9 and TRBO-G-3'gGFP in the presence of P19 also resulted in elevated levels percentages of indels (insertions and deletions). These data also revealed an age-related phenomenon in plants whereby the RNA suppressor P19 had more of an effect in older plants. Lastly, we used a single TRBO vector to express both Cas9 and a sgRNA. Taken together, we suggest that viral RNA suppressors could be used for further optimization of single viral vector delivery of CRISPR gene editing parts.

摘要

我们报告了进一步开发农杆菌浸润型烟草花叶病毒(TMV)基础过表达(TRBO)载体,将 CRISPR/Cas9 组件递送到植物中。首先,当与基因沉默抑制剂(如 TMV 126-kDa 复制酶或番茄丛矮病毒 P19 蛋白)共表达时,来自二元质粒的 Cas9(HcoCas9)蛋白的产量增加。这种抑制剂产生的 Cas9 表达水平升高转化为通过 TRBO-G-3'gGFP 表达 GFP 以及靶向 Nicotiana benthamiana GFP 表达系 16c 中的 mgfp5 基因的单向导 RNA 介导的有效基因编辑。此外,HcoCas9 编码 RNA,一个 4.2kb 的大 cargo 插入物,从 TRBO-HcoCas9 表达,再次在与 P19 共表达时以更高水平表达 Cas9 蛋白。同样,在 P19 存在下共递送 TRBO-HcoCas9 和 TRBO-G-3'gGFP 也导致插入缺失(插入和缺失)的百分比升高。这些数据还揭示了植物中与年龄相关的现象,即 RNA 抑制剂 P19 在较老的植物中具有更大的影响。最后,我们使用单个 TRBO 载体同时表达 Cas9 和 sgRNA。总之,我们认为病毒 RNA 抑制剂可用于进一步优化 CRISPR 基因编辑部件的单个病毒载体传递。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b72/7990971/a9d85aade4c5/41598_2021_85366_Fig1_HTML.jpg

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