Abstract
BackgroundThe 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.ResultsHere 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0101-2) contains supplementary material, which is available to authorized users.
Highlights
The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the clustered regularly interspaced short palindromic repeats (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
GB‐adapted cloning strategy for CRISPR/Cas9 plant constructs To facilitate the assembly of CRISPR/Cas9 constructs and the delivery of multiple guide RNAs in the same T-DNA, we designed the CRISPR cloning workflow depicted on Fig. 1a
The D/M-Target is combined with a PolIII promoter and with the scaffold RNA in a cyclic digestion/ligation Golden Gate reaction [22] to build the complete guide RNA (gRNA) expression cassette
Summary
The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas 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 clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune bacterial system has rapidly become a powerful technology for genome editing in many organisms This system is based on a guide RNA (gRNA) that directs the Streptococcus pyogenes Cas nuclease to its target site. A successful alternative for plants is the use of Agrobacterium mediated T-DNA transformation, followed by callus induction and organogenic plant regeneration (or floral dip transformation in the case of Arabidopsis) In this case, T-DNA-delivered gRNA–Cas, besides acting transiently during callus formation, can integrate in the genome and continue its activity in somatic tissues [4]. To exploit the full potential of the T-DNA strategy it is important to expand the ability to combine different gRNAs together with Cas within a single T-DNA, as it has been demonstrated that all-in-one plasmid approaches significantly increase editing efficiency [15]
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