Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene Editing
ABSTRACT Two types of CRISPR/Cas systems (Cas9 and Cas13) have been used to combat eukaryotic viruses successfully. In this study, we established resistance to the DNA virus BSCTV and RNA virus TMV in Nicotiana benthamiana using the CRISPR‐Cas12a multiplex gene editing system. We employed two effect...
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| Format: | Article |
| Language: | English |
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Wiley
2025-04-01
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| Series: | Plant Direct |
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| Online Access: | https://doi.org/10.1002/pld3.70070 |
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| author | Lili Luo Liqing Miao Xuhui Ma Jinjin Hu Suzhen Li Wenzhu Yang Shuai Ma Rumei Chen Xiaoqing Liu |
| author_facet | Lili Luo Liqing Miao Xuhui Ma Jinjin Hu Suzhen Li Wenzhu Yang Shuai Ma Rumei Chen Xiaoqing Liu |
| author_sort | Lili Luo |
| collection | DOAJ |
| description | ABSTRACT Two types of CRISPR/Cas systems (Cas9 and Cas13) have been used to combat eukaryotic viruses successfully. In this study, we established resistance to the DNA virus BSCTV and RNA virus TMV in Nicotiana benthamiana using the CRISPR‐Cas12a multiplex gene editing system. We employed two effector proteins LbCas12a and FnCas12a coupled with six guide RNAs targeting virus genome and a novel mRNA–gRNA nucleic acid complex to transport gRNA efficiently. Compared with the BSCTV accumulation in the wild‐type N. benthamiana, it was reduced by more than 90% by most transgenic events derived at 7 days post‐inoculation. Additionally, the shoot‐tip leaves were normal in the transgenic plants, whereas they appeared severely curled and stunted in wild‐type N. benthamiana at 15 days post‐infection. Target sites evaluation revealed that the editing system can directly destroy the structure of BSCTV viral genomes via large fragment deletions. We quantified TMV virus accumulation in the transgenic N. benthamiana lines by monitoring dynamic changes in GFP fluorescence and quantitative analysis by qPCR showed that the CRISPR‐Cas12a system can introduce TMV virus resistance to N. benthamiana by preventing its systemic spread. Our study provides an innovative strategy—an mRNA–gRNA nucleic acid complex—which has proven to be highly effective in the gene‐editing system and offers an efficient antiviral approach for generating virus‐resistant plants. |
| format | Article |
| id | doaj-art-c924b0b10f2e4018a26f6992040efffe |
| institution | OA Journals |
| issn | 2475-4455 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Wiley |
| record_format | Article |
| series | Plant Direct |
| spelling | doaj-art-c924b0b10f2e4018a26f6992040efffe2025-08-20T02:19:37ZengWileyPlant Direct2475-44552025-04-0194n/an/a10.1002/pld3.70070Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene EditingLili Luo0Liqing Miao1Xuhui Ma2Jinjin Hu3Suzhen Li4Wenzhu Yang5Shuai Ma6Rumei Chen7Xiaoqing Liu8Biotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing ChinaBiotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing ChinaBiotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing ChinaKey Laboratory of Crop Biology of Anhui Province Anhui Agricultural University Hefei ChinaBiotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing ChinaBiotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing ChinaBiotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing ChinaBiotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing ChinaBiotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing ChinaABSTRACT Two types of CRISPR/Cas systems (Cas9 and Cas13) have been used to combat eukaryotic viruses successfully. In this study, we established resistance to the DNA virus BSCTV and RNA virus TMV in Nicotiana benthamiana using the CRISPR‐Cas12a multiplex gene editing system. We employed two effector proteins LbCas12a and FnCas12a coupled with six guide RNAs targeting virus genome and a novel mRNA–gRNA nucleic acid complex to transport gRNA efficiently. Compared with the BSCTV accumulation in the wild‐type N. benthamiana, it was reduced by more than 90% by most transgenic events derived at 7 days post‐inoculation. Additionally, the shoot‐tip leaves were normal in the transgenic plants, whereas they appeared severely curled and stunted in wild‐type N. benthamiana at 15 days post‐infection. Target sites evaluation revealed that the editing system can directly destroy the structure of BSCTV viral genomes via large fragment deletions. We quantified TMV virus accumulation in the transgenic N. benthamiana lines by monitoring dynamic changes in GFP fluorescence and quantitative analysis by qPCR showed that the CRISPR‐Cas12a system can introduce TMV virus resistance to N. benthamiana by preventing its systemic spread. Our study provides an innovative strategy—an mRNA–gRNA nucleic acid complex—which has proven to be highly effective in the gene‐editing system and offers an efficient antiviral approach for generating virus‐resistant plants.https://doi.org/10.1002/pld3.70070BSCTVCRISPR‐Cas12amultiplex gene editingTMVvirus resistance |
| spellingShingle | Lili Luo Liqing Miao Xuhui Ma Jinjin Hu Suzhen Li Wenzhu Yang Shuai Ma Rumei Chen Xiaoqing Liu Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene Editing Plant Direct BSCTV CRISPR‐Cas12a multiplex gene editing TMV virus resistance |
| title | Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene Editing |
| title_full | Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene Editing |
| title_fullStr | Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene Editing |
| title_full_unstemmed | Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene Editing |
| title_short | Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene Editing |
| title_sort | establishing an immune system conferring dna and rna virus resistance in plants using crispr cas12a multiplex gene editing |
| topic | BSCTV CRISPR‐Cas12a multiplex gene editing TMV virus resistance |
| url | https://doi.org/10.1002/pld3.70070 |
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