Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing
文献类型： 外文期刊
作者： Luo, Yanjie 1 ; Na, Ren 2 ; Nowak, Julia S. 1 ; Qiu, Yang 3 ; Lu, Qing Shi 1 ; Yang, Chunyan 2 ; Marsolais, Frederic 1 ;
作者机构： 1.Agr & Agri Food Canada, London Res & Dev Ctr, London, ON N5V 4T3, Canada
2.Hebei Acad Agr & Forestry Sci, Inst Cereal & Oil Crops, Shijiazhuang 050031, Hebei, Peoples R China
3.Chinese Acad Agr Sci, Inst Vegetables & Flowers, Beijing 100081, Peoples R China
4.Hebei Acad Agr & Forestry Sci, Inst Millet Crops, Shijiazhuang 050031, Hebei, Peoples R China
关键词： CRISPR-Cas9; genome editing; virus vector; soybean; Golden Gate assembly
期刊名称：BMC PLANT BIOLOGY （ 影响因子：4.215； 五年影响因子：4.96 ）
ISSN： 1471-2229
年卷期： 2021 年 21 卷 1 期
页码：
收录情况： SCI
摘要： Background A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing. Results To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected. Conclusions With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.
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