Dataset Information

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.

ABSTRACT: CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

SUBMITTER: Kiryushkin AS

PROVIDER: S-EPMC8747350 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.

Kiryushkin Alexey S AS Ilina Elena L EL Guseva Elizaveta D ED Pawlowski Katharina K Demchenko Kirill N KN

Plants (Basel, Switzerland) 20211224 1

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different <i>Agrobacterium rhizogenes</i> strains that are widely ...[more]

PMID: 35009056

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Project description:The stable transformation of common bean is a challenging and time-consuming process. Although CRISPR/Cas9 has revolutionized gene editing with its high efficiency and specificity, the performance of the system can be affected by multiple factors, such as sgRNA specificity and effectiveness, and the choice of promoter used to drive Cas9 expression. The use of a hairy root transformation system to initially check the efficiency of sgRNAs and the impact of different promoters could speed up this process and increase the chances of success. We initially tested three different transformation methods to induce hairy roots and selected a preferred method suitable for a variety of different common bean genotypes. This method involved inoculating a severed radicle with Rhizobium rhizogenes K599 and was fast, had a high transformation frequency of 42-48%, and resulted in numerous hairy roots. This method was further used for the transformation of explants using R. rhizogenes harboring different CRISPR/Cas9 constructs and evaluated the on-target activity of sgRNAs targeting raffinose family oligosaccharides biosynthetic genes and the impact of different promoters driving Cas9 on the gene editing efficiency. Additionally, we evaluated the reliability of the in silico tools, CRISPOR, CRISPR RGEN, and inDelphi to predict the sgRNA efficiencies and resulting mutations. Our results showed that the hairy root transformation system allows for rapid evaluation of multiple sgRNAs and promoters. We also identified several highly efficient sgRNAs that induced frameshift mutations at rates of up to 70% when a parsley ubiquitin promoter was driving Cas9 expression, providing valuable information for the selection of the most effective sgRNAs and promoters for future transformation experiments. Although most of the computational models used to predict the sgRNA efficiency did not match the in planta results, the Lindel model proved to be the most reliable for P. vulgaris, accurately predicting the sgRNA efficiency and the type of induced mutation in most hairy roots. Furthermore, the inDelphi algorithm could correctly predict deletions and single nucleotide insertions resulting from DNA double-strand breaks in common bean. These results offer promising implications for enhancing precise editing in plants because they provide the possibility of predicting repair outcomes.

Project description:BackgroundAgrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function.ResultsIn this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58-64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg' B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein-protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean.ConclusionA rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein-protein interactions and gene editing system evaluation.

Dataset Information

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.

Publications

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.

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