Dataset Information

Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes.

ABSTRACT: Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 (CCR1), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1-edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1-edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes.

SUBMITTER: Dai Y

PROVIDER: S-EPMC7279396 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Implementing the CRISPR/Cas9 Technology in <i>Eucalyptus</i> Hairy Roots Using Wood-Related Genes.

Dai Ying Y Hu Guojian G Dupas Annabelle A Medina Luciano L Blandels Nils N Clemente Hélène San HS Ladouce Nathalie N Badawi Myriam M Hernandez-Raquet Guillermina G Mounet Fabien F Grima-Pettenati Jacqueline J Cassan-Wang Hua H

International journal of molecular sciences 20200512 10

Eucalypts are the most planted hardwoods worldwide. The availability of the <i>Eucalyptus grandis</i> genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-rela ...[more]

PMID: 32408486

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Project description:BackgroundPeanut is an important legume crop growing worldwide. With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas9 system is emerging as a robust tool for gene functional study and crop improvement, which haven't been extensively utilized in peanut yet. Peanut plant forms root nodules to fix nitrogen through a symbiotic relationship with rhizobia. In model legumes, the response of plants to rhizobia is initiated by Nod factor receptors (NFRs). However, information about the function of NFRs in peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function of NFR genes.ResultsWe firstly identified four AhNFR1 genes and two AhNFR5 genes in cultivated peanut (Tifrunner). The gene expression analysis showed that the two AhNFR1 and two AhNFR5 genes had high expression levels in nodulating (Nod+) line E5 compared with non-nodulating (Nod-) line E4 during the process of nodule formation, suggesting their roles in peanut nodulation. To further explore their functions in peanut nodulation, we applied CRISPR technology to create knock-out mutants of AhNFR1 and AhNFR5 genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selected AhNFR1 and AhNFR5 genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the two AhNFR5 genes showed Nod- phenotype, whereas mutants with editing in the two selected AhNFR1 genes could still form nodules after rhizobia inoculation.ConclusionsThis study showed that CRISPR-Cas9 could be used in peanut hairy root transformation system for peanut functional genomic studies, specifically on the gene function in roots. By using CRISPR-Cas9 targeting peanut AhNFR genes in hairy root transformation system, we validated the function of AhNFR5 genes in nodule formation in peanut.

Project description:BackgroundTraditional CRISPR/Cas9 systems that rely on U6 or U3 snRNA promoters (RNA polymerase III-dependent promoters) can only achieve constitutive gene editing in plants, hampering the functional analysis of specifically expressed genes. Ribozyme-mediated CRISPR/Cas9 systems increase the types of promoters which can be used to transcribe sgRNA. Therefore, such systems allow specific gene editing; for example, transcription of the artificial gene Ribozyme-sgRNA-Ribozyme (RGR) is initiated by an RNA polymerase II-dependent promoter. Genetic transformation is indispensable for editing plant genes. In certain plant species, including pyrethrum, genetic transformation remains challenging to do, limiting the functional verification of novel CRISPR/Cas9 systems. Thus, this study's aim was to develop a simple Agrobacterium rhizogenes-mediated hairy root transformation system to analyze the function of a ribozyme-mediated CRISPR/Cas9 system in pyrethrum.ResultsA hairy root transformation system for pyrethrum is described, with a mean transformation frequency of 7%. Transgenic hairy roots transformed with the pBI121 vector exhibited significantly increased beta-glucuronidase staining as a visual marker of transgene expression. Further, a ribozyme-based CRISPR/Cas9 vector was constructed to edit the TcEbFS gene, which catalyzes synthesis of the defense-related compound (E)-β-farnesene in pyrethrum. The vector was transferred into the hairy roots of pyrethrum and two stably transformed hairy root transgenic lines obtained. Editing of the TcEbFS gene in the hairy roots was evaluated by gene sequencing, demonstrating that both hairy root transgenic lines had DNA base loss at the editing target site. Gas chromatography-mass spectrometry showed that the (E)-β-farnesene content was significantly decreased in both hairy root transgenic lines compared with the empty vector control group. Altogether, these results show that RGR can be driven by the CaMV35S promoter to realize TcEbFS gene editing in pyrethrum hairy roots.ConclusionAn A. rhizogenes-mediated hairy root transformation and ribozyme-mediated CRISPR/Cas9 gene editing system in pyrethrum was established, thereby facilitating gene editing in specific organs or at a particular developmental stage in future pyrethrum research.

Dataset Information

Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes.

Publications

Implementing the CRISPR/Cas9 Technology in <i>Eucalyptus</i> Hairy Roots Using Wood-Related Genes.

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