Genome Engineering and Agriculture: Opportunities and Challenges.

In recent years, plant biotechnology has witnessed unprecedented technological change. Advances in high-throughput sequencing technologies have provided insight into the location and structure of functional elements within plant DNA. At the same time, improvements in genome engineering tools have enabled unprecedented control over genetic material. These technologies, combined with a growing understanding of plant systems biology, will irrevocably alter the way we create new crop varieties. As the first wave of genome-edited products emerge, we are just getting a glimpse of the immense opportunities the technology provides. We are also seeing its challenges and limitations. It is clear that genome editing will play an increased role in crop improvement and will help us to achieve food security in the coming decades; however, certain challenges and limitations must be overcome to realize the technology's full potential.

Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation.

Genome-wide association (GWA) studies offer the opportunity to identify genes that contribute to naturally occurring variation in quantitative traits. However, GWA relies exclusively on statistical association, so functional validation is necessary to make strong claims about gene function. We used a combination of gene-disruption platforms (Tnt1 retrotransposons, hairpin RNA-interference constructs, and CRISPR/Cas9 nucleases) together with randomized, well-replicated experiments to evaluate the function of genes that an earlier GWA study in Medicago truncatula had identified as candidates contributing to variation in the symbiosis between legumes and rhizobia. We evaluated ten candidate genes found in six clusters of strongly associated single nucleotide polymorphisms, selected on the basis of their strength of statistical association, proximity to annotated gene models, and root or nodule expression. We found statistically significant effects on nodule production for three candidate genes, each validated in two independent mutants. Annotated functions of these three genes suggest their contributions to quantitative variation in nodule production occur through processes not previously connected to nodulation, including phosphorous supply and salicylic acid-related defense response. These results demonstrate the utility of GWA combined with reverse mutagenesis technologies to discover and validate genes contributing to naturally occurring variation in quantitative traits. The results highlight the potential for GWA to complement forward genetics in identifying the genetic basis of ecologically and economically important traits.

High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9.

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.

Direct stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oil.

BACKGROUND: The ability to modulate levels of individual fatty acids within soybean oil has potential to increase shelf-life and frying stability and to improve nutritional characteristics. Commodity soybean oil contains high levels of polyunsaturated linoleic and linolenic acid, which contribute to oxidative instability - a problem that has been addressed through partial hydrogenation. However, partial hydrogenation increases levels of trans-fatty acids, which have been associated with cardiovascular disease. Previously, we generated soybean lines with knockout mutations within fatty acid desaturase 2-1A (FAD2-1A) and FAD2-1B genes, resulting in oil with increased levels of monounsaturated oleic acid (18:1) and decreased levels of linoleic (18:2) and linolenic acid (18:3). Here, we stack mutations within FAD2-1A and FAD2-1B with mutations in fatty acid desaturase 3A (FAD3A) to further decrease levels of linolenic acid. Mutations were introduced into FAD3A by directly delivering TALENs into fad2-1a fad2-1b soybean plants. RESULTS: Oil from fad2-1a fad2-1b fad3a plants had significantly lower levels of linolenic acid (2.5 %), as compared to fad2-1a fad2-1b plants (4.7 %). Furthermore, oil had significantly lower levels of linoleic acid (2.7 % compared to 5.1 %) and significantly higher levels of oleic acid (82.2 % compared to 77.5 %). Transgene-free fad2-1a fad2-1b fad3a soybean lines were identified. CONCLUSIONS: The methods presented here provide an efficient means for using sequence-specific nucleases to stack quality traits in soybean. The resulting product comprised oleic acid levels above 80 % and linoleic and linolenic acid levels below 3 %.

Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases.

Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species.

Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA.

Plant genome engineering using sequence-specific nucleases (SSNs) promises to advance basic and applied plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the plant genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to plant cells, namely transformation of messenger RNA (mRNA) encoding TAL effector nucleases (TALENs). mRNA delivery of a TALEN pair targeting the Nicotiana benthamiana ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were <10bp; in contrast, 88% of insertions generated through DNA delivery were >10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5' and 3' untranslated regions (UTRs) from Arabidopsis thaliana genes to the TALEN coding sequence. UTRs from an A. thaliana adenine nucleotide α hydrolases-like gene (At1G09740) enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of genome editing.

Improving cold storage and processing traits in potato through targeted gene knockout.

Cold storage of potato tubers is commonly used to reduce sprouting and extend postharvest shelf life. However, cold temperature stimulates the accumulation of reducing sugars in potato tubers. Upon high-temperature processing, these reducing sugars react with free amino acids, resulting in brown, bitter-tasting products and elevated levels of acrylamide--a potential carcinogen. To minimize the accumulation of reducing sugars, RNA interference (RNAi) technology was used to silence the vacuolar invertase gene (VInv), which encodes a protein that breaks down sucrose to glucose and fructose. Because RNAi often results in incomplete gene silencing and requires the plant to be transgenic, here we used transcription activator-like effector nucleases (TALENs) to knockout VInv within the commercial potato variety, Ranger Russet. We isolated 18 plants containing mutations in at least one VInv allele, and five of these plants had mutations in all VInv alleles. Tubers from full VInv-knockout plants had undetectable levels of reducing sugars, and processed chips contained reduced levels of acrylamide and were lightly coloured. Furthermore, seven of the 18 modified plant lines appeared to contain no TALEN DNA insertions in the potato genome. These results provide a framework for using TALENs to quickly improve traits in commercially relevant autotetraploid potato lines.

High-frequency, precise modification of the tomato genome.

BACKGROUND: The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA. RESULTS: Here, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences. CONCLUSIONS: High-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA.

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.

The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.

Enabling plant synthetic biology through genome engineering.

Synthetic biology seeks to create new biological systems, including user-designed plants and plant cells. These systems can be employed for a variety of purposes, ranging from producing compounds of industrial or therapeutic value, to reducing crop losses by altering cellular responses to pathogens or climate change. To realize the full potential of plant synthetic biology, techniques are required that provide control over the genetic code - enabling targeted modifications to DNA sequences within living plant cells. Such control is now within reach owing to recent advances in the use of sequence-specific nucleases to precisely engineer genomes. We discuss here the enormous potential provided by genome engineering for plant synthetic biology.

Conferring resistance to geminiviruses with the CRISPR-Cas prokaryotic immune system.

To reduce crop losses due to geminivirus infection, we targeted the bean yellow dwarf virus (BeYDV) genome for destruction with the CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR-associated proteins) system. Transient assays using BeYDV-based replicons revealed that CRISPR-Cas reagents introduced mutations within the viral genome and reduced virus copy number. Transgenic plants expressing CRISPR-Cas reagents and challenged with BeYDV had reduced virus load and symptoms, thereby demonstrating a novel strategy for engineering resistance to geminiviruses.

DNA replicons for plant genome engineering.

Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of plant genomes remains challenging due to ineffective methods for delivering reagents for genome engineering to plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activator-like effector nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA. In addition to the nuclease-mediated DNA double-strand breaks, gene targeting was promoted by replication of the repair template and pleiotropic activity of the geminivirus replication initiator proteins. We demonstrate the feasibility of using geminivirus replicons to generate plants with a desired DNA sequence modification. By adopting a general plant transformation method, plantlets with a desired DNA change were regenerated in <6 weeks. These results, in addition to the large host range of geminiviruses, advocate the use of replicons for plant genome engineering.

Tailor-made mutations in Arabidopsis using zinc finger nucleases.

Zinc finger nucleases (ZFNs) are proteins engineered to make site-specific double-strand breaks (DSBs) in a DNA sequence of interest. Imprecise repair of the ZFN-induced DSBs by the nonhomologous end-joining (NHEJ) pathway results in a spectrum of mutations, such as nucleotide substitutions, insertions, and deletions. Here we describe a method for targeted mutagenesis in Arabidopsis with ZFNs, which are engineered by context-dependent assembly (CoDA). This ZFN-induced mutagenesis method is an alternative to other currently available gene knockout or knockdown technologies and is useful for reverse genetic studies.

Targeted mutagenesis for functional analysis of gene duplication in legumes.

Assessment of gene function oftentimes requires mutant populations that can be screened by forward or reverse genetic analysis. The situation becomes more complicated in polyploidy or paleopolyploid genomes that have two or more copies for most genes. Here we describe a method for engineering zinc-finger nucleases (ZFNs) for the purpose of creating targeted mutations in the paleopolyploid soybean genome. ZFNs are recombinant proteins composed of an engineered zinc-finger array fused to a nonspecific cleavage domain. When engineered to recognize a specific nucleotide sequence, the cleavage domain will generate highly mutagenic DNA double-strand breaks frequently resulting in insertions and deletions at the target locus. Depending on the number of target sites present within the genome, this method has the capacity to target either single- or multi-copy gene families. In this chapter, we describe an inexpensive, rapid, and user-friendly approach for ZFN assembly and application in soybean based on the previously described context-dependent assembly method.

Targeted deletion and inversion of tandemly arrayed genes in Arabidopsis thaliana using zinc finger nucleases.

Tandemly arrayed genes (TAGs) or gene clusters are prevalent in higher eukaryotic genomes. For example, approximately 17% of genes are organized in tandem in the model plant Arabidopsis thaliana. The genetic redundancy created by TAGs presents a challenge for reverse genetics. As molecular scissors, engineered zinc finger nucleases (ZFNs) make DNA double-strand breaks in a sequence-specific manner. ZFNs thus provide a means to delete TAGs by creating two double-strand breaks in the gene cluster. Using engineered ZFNs, we successfully targeted seven genes from three TAGs on two Arabidopsis chromosomes, including the well-known RPP4 gene cluster, which contains eight resistance (R) genes. The resulting gene cluster deletions ranged from a few kb to 55 kb with frequencies approximating 1% in somatic cells. We also obtained large chromosomal deletions of ~9 Mb at approximately one tenth the frequency, and gene cluster inversions and duplications also were achieved. This study demonstrates the ability to use sequence-specific nucleases in plants to make targeted chromosome rearrangements and create novel chimeric genes for reverse genetics and biotechnology.

Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases.

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform--a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting dicer-like (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.

Cryptococcal cell morphology affects host cell interactions and pathogenicity.

Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 microm. Cell enlargement was observed in vivo, producing cells up to 100 microm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aDelta pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.