Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an ever-growing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.

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. 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. 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.

Summary: The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA‐guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB‐assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target‐dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on‐target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a‐free segregating T2 lines to assess possible off‐target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of −10 to −2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off‐target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.

SUMMARY: Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR‐associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR‐Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target‐specific and a screening process is usually required to identify those most effective. Plant virus‐derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus‐induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX‐based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus‐free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR‐Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.

Synthetic Biology (SynBio) aims at rewiring plant metabolic and developmental programs with orthogonal regulatory circuits. This endeavour requires new molecular tools able to interact with endogenous factors in a potent yet at the same time highly specific manner. A promising new class of SynBio tools that could play this function are the synthetic transcriptional activators based on CRISPR/Cas9 architecture, which combine autonomous transcriptional activation domains (TADs) capable of recruiting the cellular transcription machinery, with the easily customizable DNA‐binding activity of nuclease‐inactivated Cas9 protein (dCas9), creating so‐called Programmable Transcriptional Activators (PTAs). Initial dCas9‐PTAs versions comprising single domain fusions to Cas9 showed only low/moderate activation rates in plants. A new generation PTAs developed for mammalian cells combine several TADs attached to a single dCas9 protein, reaching higher activation rates. Multi‐TAD display can be achieved following different strategies (see Figure 1b). The SunTag strategy (Tanenbaum et al. 2014) uses multi‐epitope tags to attach multiple TADs to the dCas9 protein, whereas SAM and scRNA strategies (Mali et al. 2013, Konermann et al. 2015) use RNA aptamers added to the gRNA scaffold as secondary anchoring sites for TADs. Recently, some multi‐TAD designs have been successfully applied to plants (Li et al. 2017, Lowder et al. 2018), however a comprehensive multi‐parallel test for plant dCas9‐PTAs including multi‐TAD designs is still missing, leaving the door open for design improvements. Here we show a systematic comparison of 43 SunTag, SAM and scRNA‐based TAD combinations tested for their ability to activate different promoters fused to a Luciferase reporter.

Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation. To facilitate the handling of functional descriptions, we developed a new version (v3.0) of the GoldenBraid (GB) webtool that integrates the experimental data and displays it in the form of datasheets. We report the use of the Luciferase/Renilla (Luc/Ren) transient agroinfiltration assay in Nicotiana benthamiana as a standard to estimate relative transcriptional activities conferred by regulatory phytobricks, and show the consistency and reproducibility of this method in the characterization of a synthetic phytobrick based on the CaMV35S promoter. Furthermore, we illustrate the potential for combinatorial optimization and incremental innovation of the GB3.0 platform in two separate examples, (i) the development of a collection of orthogonal transcriptional regulators based on phiC31 integrase and (ii) the design of a small genetic circuit that connects a glucocorticoid switch to a MYB/bHLH transcriptional activation module.