Development of a Csy4-Processing TRV-Based CRISPR/Cas9 Genome Editing System in Nicotiana benthamiana

文献类型： 外文期刊

第一作者： Luo, Yanjie

作者： Luo, Yanjie;Na, Ren;Tang, Xiaodong;Yu, Cuihong;Na, Ren;Qiu, Yang;Nowak, Julia S.;Lu, Qing Shi;Tian, Lining;Luo, Yanjie

作者机构：

关键词： CRISPR/Cas9; Csy4 nuclease; Genome editing; Viral vector; TRV

期刊名称：PLANT MOLECULAR BIOLOGY REPORTER （ 影响因子：2.1； 五年影响因子：2.0 ）

ISSN： 0735-9640

年卷期： 2024 年

页码：

收录情况： SCI

摘要： The CRISPR/Cas9 system is a site-specific genome editing tool that has been widely used in various plant species. The plant virus-based gRNA (guide RNA) delivery system, which differs from the typical Agrobacterium-mediated transformation, is an attractive method to facilitate the application of CRISPR/Cas9. The virally delivered gRNA is usually driven by heterologous plant U6 or viral promoters (e.g., pea early-browning virus, PEBV; barley stripe mosaic virus, BSMV). However, heterologous promoters may have poor performance in some cases. In this paper, a feasible option to detach gRNA(s) from the virus genome is employed. Specifically, the Csy4-RNA processing system is used to release gRNA(s) from the tobacco rattle virus (TRV). The coding sequences of Cas9 and Csy4 nucleases are cloned into a single polycistronic expression cassette under an estrogen-inducible promoter in a binary vector, and the gRNA is cloned into the TRV genome flanked by two 20 bp Csy4 recognition sites. The results show that the Csy4-processing TRV-based delivery system works effectively in targeting single and multiple sites, nucleotide replacement, and large fragment deletion in Cas9-mediated genome editing via transient expression in Nicotiana benthamiana. The Csy4-TRV is a promising gRNA(s) processing and delivery system for CRISPR/Cas9 genome editing. This method can be easily adapted to other plant RNA viruses, facilitating the application of the CRISPR/Cas9 system in plants.

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