High-efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus

Nagalakshmi, Ugrappa; Meier, Nathan; Liu, Jau-Yi; Voytas, Daniel F.; Dinesh‐Kumar, Savithramma P.

doi:10.1093/plphys/kiac159

Cited by 31 publications

(35 citation statements)

References 12 publications

(14 reference statements)

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“…Using both co-delivery of the two spilt-Cas9 proteins and BiFC technology, the results of gene-editing and BiFC indicated that protein-protein interactions in cotton were successfully performed (Figures 5D and 6). For the BiFC, although we detected the yellow fluorescence signals in the cotyledons, we suspect that the signals would also be detected at the 4-6 true leaves stage because of the mobility of the virus [42]. Notably, we mixed the virus homogenate in equal proportions instead of directly co-injecting Agrobacterium containing CRISPR editing elements into N. benthamiana (Figure 6C).…”

Section: The Bsmv System Can Be Successfully Used For Gene Function S...mentioning

confidence: 99%

A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus

Chen

Huang

Luo

et al. 2022

Plants

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The difficulty of genetic transformation has restricted research on functional genomics in cotton. Thus, a rapid and efficient method for gene overexpression that does not rely on genetic transformation is needed. Virus-based vectors offer a reasonable alternative for protein expression, as viruses can infect the host systemically to achieve expression and replication without transgene integration. Previously, a novel four-component Barley stripe mosaic virus (BSMV) was reported to overexpress large fragments of target genes in plants over a long period of time, which greatly simplified the study of gene overexpression. However, whether this system can infect cotton and stably overexpress target genes has not yet been studied. In this study, we verified that this new BSMV system can infect cotton through seed imbibition and systemically overexpress large fragments of genes (up to 2340 bp) in cotton. The target gene that was fused with GFP was expressed at a high level in the roots, stems, and cotyledons of cotton seedlings, and stable fluorescence signals were detected in the cotton roots and leaves even after 4 weeks. Based on the BSMV overexpression system, the subcellular localization marker line of endogenous proteins localized in the nucleus, endoplasmic reticulum, plasma membrane, Golgi body, mitochondria, peroxisomes, tonoplast, and plastids were quickly established. The overexpression of a cotton Bile Acid Sodium Symporter GhBASS5 using the BSMV system indicated that GhBASS5 negatively regulated salt tolerance in cotton by transporting Na+ from underground to the shoots. Furthermore, multiple proteins were co-delivered, enabling co-localization and the study of protein–protein interactions through co-transformation. We also confirmed that the BSMV system can be used to conduct DNA-free gene editing in cotton by delivering split-SpCas9/sgRNA. Ultimately, the present work demonstrated that this BSMV system could be used as an efficient overexpression system for future cotton gene function research.

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Section: The Bsmv System Can Be Successfully Used For Gene Function S...mentioning

confidence: 99%

A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus

Chen

Huang

Luo

et al. 2022

Plants

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“…Transgenic Arabidopsis plants expressing base-editing reagents are often somatic mosaics in the first generation, and multiple generations are required to fix edited alleles ( Kang et al, 2018 ; Li et al, 2019 ; Choi et al, 2021 ). Recently, heritable targeted mutagenesis has been achieved using RNA or DNA viruses to deliver single guide RNAs (sgRNAs) to the germline of Cas9-expressing transgenic plants ( Ellison et al, 2020 ; Lei et al, 2021 ; Li et al, 2021 ; Nagalakshmi et al, 2022 ). Here, we show that this approach can be applied to base-editing using RNA viral vectors to deliver sgRNAs to transgenic Arabidopsis lines expressing a cytidine deaminase base-editor ( Supplemental Figure S1, A and B ).…”

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confidence: 99%

“…Prior to cloning into Tobacco Rattle Virus (TRV) vectors, the esgRNAs were fused at their 3′-ends to a tRNA isoleucine (tRNA Ileu ). We and others have previously shown that tRNAs and other RNAs that promote cell-to-cell movement can increase virus-mediated mutagenesis when fused to sgRNAs ( Ellison et al, 2020 ; Lei et al, 2021 ; Nagalakshmi et al, 2022 ). The TRV vectors were delivered to the transgenic L8 line via the Agrobacterium tumefaciens flooding method ( Nagalakshmi et al, 2022 ).…”

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confidence: 99%

“…We and others have previously shown that tRNAs and other RNAs that promote cell-to-cell movement can increase virus-mediated mutagenesis when fused to sgRNAs ( Ellison et al, 2020 ; Lei et al, 2021 ; Nagalakshmi et al, 2022 ). The TRV vectors were delivered to the transgenic L8 line via the Agrobacterium tumefaciens flooding method ( Nagalakshmi et al, 2022 ). The infected plants were grown under a 24°C/12-h-light and 22°C/12-h-dark cycle.…”

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confidence: 99%

“…The high number of homozygous mutants recovered may be because they grow better on the C17 selection medium relative to heterozygous and mosaic plants. We would like to note that in a companion study, TRV was used to deliver a PDS sgRNA to transgenic Arabidopsis plants expressing Cas9 ( Nagalakshmi et al, 2022 ). Mutagenesis was assessed in seeds produced at different times during development (early, middle, and late stages).…”

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confidence: 99%

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