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| Screening of Arabidopsis thaliana Cas9-expressing Lines with Enhanced VIGE-induced Target Gene Mutations |
| PENG Jing*, MAIERHABA·Hai-San*, CHEN Lin-Yang, KAMIRAN·Mai-Tu-Song, LIN Jia-Hao, LIU Zhuang, LEI Jian-Feng** |
| College of Agronomy/Key Laboratory of Agricultural Biotechnology, Xinjiang Agricultural University, Urumqi 830052, China |
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Abstract Plant virus-mediated gene editing (VIGE) system can rapidly screen high-efficiency sgRNA (single guide RNA) for targeted editing of plant genes, providing a gene editing tool for the targeted creation of genetic mutant materials. In order to screen the VIGE receptor materials capable of efficiently knockout the target genes in Arabidopsis thaliana, this study using the AtBRI1 (brassinosteroid insensitive 1) and AtGL2 (GLABRA 2) as target genes, and explored the editing efficiency of different tissue-specific Cas9 overexpression (Cas9-OE) lines as VIGE receptors for these 2 genes based on the Cotton leaf crumple virus (CLCrV)-mediated VIGE system. The expression of Cas9 gene in different tissue-specific Cas9-OE lines was examined by qPCR, and it was found that Cas9 gene was stably genetically expressed in different tissue-specific Cas9-OE lines. The leaves of different tissue-specific Cas9-OE plants were inoculated with CLCrV- AtU6-26:: AtBRI1-sgRNA and CLCrV-AtU6-26:: AtGL2-sgRNA by Agrobacterium-mediated transient transformation. Mutation detection results showed that the 3 tissue-specific Cas9-OE lines as VIGE receptors could achieve targeted editing of AtBRI1 and AtGL2 genes, and different base insertions, substitutions and deletion mutation types appeared in the target sequence regions of these 2 genes, demonstrating the effectiveness of these 3 tissue-specific Cas9-OE lines as VIGE receptors. Further mutation detection was performed on each individual plant inoculated with CLCrV-AtU6-26:: AtBRI1-sgRNA and CLCrV-AtU6-26::AtGL2-sgRNA. The mutation analysis showed that ProCDC45:: Cas9 and ProYao:: Cas9 had relatively high editing efficiency for AtBRI1 and AtGL2 genes with editing efficiency of 50%~81.25%, which proved that ProCDC45:: Cas9 and ProYao:: Cas9 transgenic Arabidopsis can be used as ideal VIGE receptors for gene editing research in Arabidopsis. The study provides a basis for further improving the gene editing efficiency of the VIGE system in Arabidopsis and efficiently creating Arabidopsis mutant materials.
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Received: 31 July 2022
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Corresponding Authors:
**kyleijianfeng@163.com
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| About author:: *These authors contributed equally to this work |
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