Dataset Information

A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard.

ABSTRACT: BACKGROUND:The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell. The engineering principles of standardization and modularity applied to DNA cloning are impacting plant genetic engineering, by increasing multigene assembly efficiency and by fostering the exchange of well-defined physical DNA parts with precise functional information. RESULTS:Here we describe the adaptation of the RNA-guided Cas9 system to GoldenBraid (GB), a modular DNA construction framework being increasingly used in Plant Synthetic Biology. In this work, the genetic elements required for CRISPRs-based editing and transcriptional regulation were adapted to GB, and a workflow for gRNAs construction was designed and optimized. New software tools specific for CRISPRs assembly were created and incorporated to the public GB resources site. CONCLUSIONS:The functionality and the efficiency of gRNA-Cas9 GB tools were demonstrated in Nicotiana benthamiana using transient expression assays both for gene targeted mutations and for transcriptional regulation. The availability of gRNA-Cas9 GB toolbox will facilitate the application of CRISPR/Cas9 technology to plant genome engineering.

SUBMITTER: Vazquez-Vilar M

PROVIDER: S-EPMC4736081 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard.

Vazquez-Vilar Marta M Bernabé-Orts Joan Miquel JM Fernandez-Del-Carmen Asun A Ziarsolo Pello P Blanca Jose J Granell Antonio A Orzaez Diego D

Plant methods 20160201

<h4>Background</h4>The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell. The engineering principles of standardization and modularity applied to DNA cloning are impacting plant genetic en ...[more]

PMID: 26839579

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Project description:We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated "Edit Plasmids," were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions at one of the Cas9 cleavage sites in Chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated non-homologous end joining (NHEJ) repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.

Dataset Information

A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard.

Publications

A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard.

Similar Datasets

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