Heritable, multinucleotide deletions in plants using viral delivery of a repair exonuclease and guide RNAs.

CRISPR/Cas9-mediated mutagenesis typically results in short insertion/deletion mutations, which are often too small to disrupt the function of cis-acting regulatory elements. Here, we describe a highly efficient in planta gene editing approach called VirTREX2-HLDel that achieves heritable multinucleotide deletions in both protein-coding genes and noncoding DNA regulatory elements. VirTREX2-HLDel uses RNA viruses to deliver both the 3 prime repair exonuclease 2 (TREX2) and single-guide RNAs. Our method enables recovery of multiplexed heritable deletions and increases the heritable gene editing frequency at poorly edited sites. We identified functional conservation and divergence of MICRORNA164 (miR164) in Nicotiana benthamiana and tomato (Solanum lycopersicum) using VirTREX2-HLDel and observed previously uncharacterized phenotypes in plants with large deletions at this locus. Our viral delivery method reduces the need for tissue culture and will accelerate the understanding of protein-coding and regulatory regions in plants.

The YABBY gene SHATTERING1 controls activation rather than patterning of the abscission zone in Setaria viridis.

Abscission is predetermined in specialized cell layers called the abscission zone (AZ) and activated by developmental or environmental signals. In the grass family, most identified AZ genes regulate AZ anatomy, which differs among lineages. A YABBY transcription factor, SHATTERING1 (SH1), is a domestication gene regulating abscission in multiple cereals, including rice and Setaria. In rice, SH1 inhibits lignification specifically in the AZ. However, the AZ of Setaria is nonlignified throughout, raising the question of how SH1 functions in species without lignification. Crispr-Cas9 knockout mutants of SH1 were generated in Setaria viridis and characterized with histology, cell wall and auxin immunofluorescence, transmission electron microscopy, hormonal treatment and RNA-Seq analysis. The sh1 mutant lacks shattering, as expected. No differences in cell anatomy or cell wall components including lignin were observed between sh1 and the wild-type (WT) until abscission occurs. Chloroplasts degenerated in the AZ of WT before abscission, but degeneration was suppressed by auxin treatment. Auxin distribution and expression of auxin-related genes differed between WT and sh1, with the signal of an antibody to auxin detected in the sh1 chloroplast. SH1 in Setaria is required for activation of abscission through auxin signaling, which is not reported in other grass species.

An extensible vector toolkit and parts library for advanced engineering of plant genomes.

Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual-luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

Direct delivery and fast-treated Agrobacterium co-culture (Fast-TrACC) plant transformation methods for Nicotiana benthamiana.

There is an expanding need to modify plant genomes to create new plant germplasm that advances both basic and applied plant research. Most current methods for plant genome modification involve regenerating plants from genetically modified cells in tissue culture, which is technically challenging, expensive and time consuming, and works with limited plant species or genotypes. Herein, we describe two Agrobacterium-based methods for creating genetic modifications on either sterilely grown or soil-grown Nicotiana benthamiana plants. These methods use developmental regulators (DRs), gene products that influence cell division and differentiation, to induce de novo meristems. Genome editing reagents, such as the RNA-guided endonuclease Cas9, may be co-delivered with the DRs to create shoots that transmit edits to the next generation. One method, called fast-treated Agrobacterium co-culture (Fast-TrACC), delivers DRs to seedlings grown aseptically; meristems that produce shoots and ultimately whole plants are induced. The other approach, called direct delivery (DD), involves delivering DRs to soil-grown plants from which existing meristems have been removed; the DRs promote the formation of new shoots at the wound site. With either approach, if transgene cassettes and/or gene editing reagents are provided, these induced, de novo meristems may be transgenic, edited or both. These two methods offer alternative approaches for generating novel plant germplasm that are cheaper and less technically challenging and take less time than standard approaches. The whole procedure from transfer DNA (T-DNA) assembly to recovery of edited plants can be completed in ~70 d for both DD and Fast-TrACC.

Analyzing Plant Gene Targeting Outcomes and Conversion Tracts with Nanopore Sequencing.

The high-throughput molecular analysis of gene targeting (GT) events is made technically challenging by the residual presetabce of donor molecules. Large donor molecules restrict primer placement, resulting in long amplicons that cannot be readily analyzed using standard NGS pipelines or qPCR-based approaches such as ddPCR. In plants, removal of excess donor is time and resource intensive, often requiring plant regeneration and weeks to months of effort. Here, we utilized Oxford Nanopore Amplicon Sequencing (ONAS) to bypass the limitations imposed by donor molecules with 1 kb of homology to the target and dissected GT outcomes at three loci in Nicotiana benthamia leaves. We developed a novel bioinformatic pipeline, Phased ANalysis of Genome Editing Amplicons (PANGEA), to reduce the effect of ONAS error on amplicon analysis and captured tens of thousands of somatic plant GT events. Additionally, PANGEA allowed us to collect thousands of GT conversion tracts 5 days after reagent delivery with no selection, revealing that most events utilized tracts less than 100 bp in length when incorporating an 18 bp or 3 bp insertion. These data demonstrate the usefulness of ONAS and PANGEA for plant GT analysis and provide a mechanistic basis for future plant GT optimization.

Fast-TrACC: A Rapid Method for Delivering and Testing Gene Editing Reagents in Somatic Plant Cells.

The production of transgenic or gene edited plants requires considerable time and effort. It is of value to know at the onset of a project whether the transgenes or gene editing reagents are functioning as predicted. To test molecular reagents transiently, we implemented an improved, Agrobacterium tumefaciens-based co-culture method called Fast-TrACC (Fast Treated Agrobacterium Co-Culture). Fast-TrACC delivers reagents to seedlings, allowing high throughput, and uses a luciferase reporter to monitor and calibrate the efficiency of reagent delivery. We demonstrate the use of Fast-TrACC in multiple solanaceous species and apply the method to test promoter activity and the effectiveness of gene editing reagents.

RNA Viral Vectors for Accelerating Plant Synthetic Biology.

The tools of synthetic biology have enormous potential to help us uncover the fundamental mechanisms controlling development and metabolism in plants. However, their effective utilization typically requires transgenesis, which is plagued by long timescales and high costs. In this review we explore how transgenesis can be minimized by delivering foreign genetic material to plants with systemically mobile and persistent vectors based on RNA viruses. We examine the progress that has been made thus far and highlight the hurdles that need to be overcome and some potential strategies to do so. We conclude with a discussion of biocontainment mechanisms to ensure these vectors can be used safely as well as how these vectors might expand the accessibility of plant synthetic biology techniques. RNA vectors stand poised to revolutionize plant synthetic biology by making genetic manipulation of plants cheaper and easier to deploy, as well as by accelerating experimental timescales from years to weeks.

Attaining the promise of plant gene editing at scale.

Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible.

VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype.

Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work, we developed a technology called VipariNama (ViN) in which vectors based on the tobacco rattle virus are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, ViN accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis.

In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.

Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs.

An in planta gene editing approach was developed wherein Cas9 transgenic plants are infected with an RNA virus that expresses single guide RNAs (sgRNAs). The sgRNAs are augmented with sequences that promote cell-to-cell mobility. Mutant progeny are recovered in the next generation at frequencies ranging from 65 to 100%; up to 30% of progeny derived from plants infected with a virus expressing three sgRNAs have mutations in all three targeted loci.

Building customizable auto-luminescent luciferase-based reporters in plants.

Bioluminescence is a powerful biological signal that scientists have repurposed as a reporter for gene expression in plants and animals. However, there are downsides associated with the need to provide a substrate to these reporters, including its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP) in planta using a composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP in planta to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for future construction of programmable auto-luminescent plant traits, such as light driven plant-pollinator interactions or light emitting plant-based sensors.

Many animals have evolved the capacity to produce light from chemical reactions. For example, an enzyme known as luciferase in fireflies produces light by acting on a molecule called luciferin. Scientists have identified the enzymes that drive several of these systems and used them to build reporters that can study the activity of genes in the tissues of plants and other lifeforms over space and time. However, these reporters often require chemicals to be added to the tissues to produce light. These chemicals tend to be expensive and may not penetrate evenly into the tissues of interest, limiting the potential applications of the reporters in research studies. Recently, it has been discovered that fungi have a bioluminescence pathway that converts a molecule known as caffeic acid into luciferin. Caffeic acid is a common molecule in plants, therefore, it is possible the fungal bioluminescence pathway could be used to build reporters that produce light without needing the addition of chemicals. Now, Khakhar et al. have inserted the genes that encode the enzymes of the fungal bioluminescence pathway into tobacco plants. The experiments found that this was sufficient to turn caffeic acid into molecules of luciferin which are able to produce light. Inserting the same genes into several other plant species, including tomatoes and dahlias, produced similar results. Further experiments showed that the fungal bioluminescence pathway can be used to build reporters that monitor the activity of plant genes throughout living tissues and over a period of several days as well as examine the response to plant hormones. Alongside studying the activities of genes in plants, Khakhar et al. propose that the toolkit developed in this work could be used to generate plants with luminescence that can be switched on or off as desired. This could have many uses including helping plants attract insects to pollinate flowers and building plant biosensors that emit light in response to environmental signals.

Overcoming bottlenecks in plant gene editing.

Agriculture has reached a technological inflection point. The development of novel gene editing tools and methods for their delivery to plant cells promises to increase genome malleability and transform plant biology. Whereas gene editing is capable of making a myriad of DNA sequence modifications, its widespread adoption has been hindered by a number of factors, particularly inefficiencies in creating precise DNA sequence modifications and ineffective methods for delivering gene editing reagents to plant cells. Here, we briefly overview the principles of plant genome editing and highlight a subset of the most recent advances that promise to overcome current limitations.

Correction: Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats.

[This corrects the article DOI: 10.1371/journal.pone.0164206.].

Plant gene editing through de novo induction of meristems.

Plant gene editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to generate gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene-editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant gene editing.