Dataset Information

First-generation genome editing in potato using hairy root transformation.

ABSTRACT: Genome editing and cis-gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T-DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%-98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%-30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ-line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.

SUBMITTER: Butler NM

PROVIDER: S-EPMC7589382 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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First-generation genome editing in potato using hairy root transformation.

Butler Nathaniel M NM Jansky Shelley H SH Jiang Jiming J

Plant biotechnology journal 20200416 11

Genome editing and cis-gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating t ...[more]

PMID: 32170801

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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.

Project description:A protocol for the induction of site-directed deletions and insertions in the genome of Brassica carinata with CRISPR is described. The construct containing the Cas9 nuclease and the guide RNA (gRNA) was delivered by the hairy root transformation technique, and a successful transformation was monitored by GFP fluorescence. PAGE analysis of an amplified region, presumably containing the deletions and insertions, demonstrated up to seven different indels in one transgenic root and in all analyzed roots a wildtype allele of the modified gene was not detectable. Interestingly, many of these mutations consisted of relatively large indels with up to 112 bp. The exact size of the deletions was determined to allow an estimation whether the targeted gene was not functional due to a considerable deletion or a frame shift within the open reading frame. This allowed a direct phenotypic assessment of the previously characterized roots and, in fact, deletions in FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 1 (BcFLA1)-a gene with an expression pattern consistent with a role in root hair architecture-resulted in shorter root hairs compared to control roots ectopically expressing an allele of the gene that cannot be targeted by the gRNA in parallel to the CRISPR construct. As an additional line of evidence, we monitored BcFLA1 expression with qPCR and detected a significant reduction of the transcript in roots with an active CRISPR construct compared to the control, although residual amounts of the transcript were detected, possibly due to inefficient nonsense-mediated mRNA decay. Additionally, the presence of deletions and insertions were verified by Sanger sequencing of the respective amplicons. In summary we demonstrate the successful application of CRISPR/Cas9 in hairy roots of B. carinata, the proof of its effectiveness and its effect on the root hair phenotype. This study paves the way for experimental strategies involving the phenotypic assessment of gene lesions by CRISPR which do not require germline transmission.

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.

Dataset Information

First-generation genome editing in potato using hairy root transformation.

Publications

First-generation genome editing in potato using hairy root transformation.

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