CRISPR/LbCas12a-Mediated Genome Editing in Soybean.

Currently methods for generating soybean edited lines are time-consuming, inefficient, and limited to certain genotypes. Here we describe a fast and highly efficient genome editing method based on CRISPR-Cas12a nuclease system in soybean. The method uses Agrobacterium-mediated transformation to deliver editing constructs and uses aadA or ALS genes as selectable marker. It only takes about 45 days to obtain greenhouse-ready edited plants at higher than 30% transformation efficiency and 50% editing rate. The method is applicable to other selectable markers including EPSPS and has low transgene chimera rate. The method is also genotype-flexible and has been applied to genome editing of several elite soybean varieties.

Automated, High-Throughput Protoplast Transfection for Gene Editing and Transgene Expression Studies.

In an era of cost-efficient gene synthesis and high-throughput construct assembly, the onus of scientific experimentation is on the rate of in vivo testing for the identification of top performing candidates or designs. Assay platforms that are relevant to the species of interest and in the tissue of choice are highly desirable. A protoplast isolation and transfection method that is compatible with a large repertoire of species and tissues would be the platform of choice. A necessary aspect of this high-throughput screening approach is the need to handle many delicate protoplast samples at the same time, which is a bottleneck for manual operation. Such bottlenecks can be mitigated with the use of automated liquid handlers for the execution of protoplast transfection steps. The method described within this chapter utilizes a 96-well head for simultaneous, high-throughput initiation of transfection. While initially developed and optimized for use with etiolated maize leaf protoplasts, the automated protocol has also been demonstrated to be compatible with other established protoplast systems, such as soybean immature embryo derived protoplast, similarly described within. This chapter also includes instructions for a sample randomization design to reduce the impact of edge effects, which might be present when microplates are used for fluorescence readout following transfection. We also describe a streamlined, expedient, and cost-effective protocol for determining gene editing efficiencies using the T7E1 endonuclease cleavage assay with a publicly available image analysis tool.

CW198 acts as a genetic insulator to block enhancer-promoter interaction in plants.

Insulators in vertebrates play a role in genome architecture and orchestrate temporo-spatial enhancer-promoter interactions. In plants, insulators and their associated binding factors have not been documented as of yet, largely as a result of a lack of characterized insulators. In this study, we took a comprehensive strategy to identify and validate the enhancer-blocking insulator CW198. We show that a 1.08-kb CW198 fragment from Arabidopsis can, when interposed between an enhancer and a promoter, efficiently abrogate the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and tobacco plants. In plants, both transcriptional crosstalk and spreading of histone modifications were rarely detectable across CW198, which resembles the insulation property observed across the CTCF insulator in the mammalian genome. Taken together, our findings support that CW198 acts as an enhancer-blocking insulator in both Arabidopsis and tobacco. The significance of the present findings and their relevance to the mitigation of mutual interference between enhancers and promoters, as well as multiple promoters in transgenes, is discussed.

Rice RS2-9, which is bound by transcription factor OSH1, blocks enhancer-promoter interactions in plants.

Insulators characterized in Drosophila and mammals have been shown to play a key role in the restriction of promiscuous enhancer-promoter interactions, as well as reshaping the topological landscape of chromosomes. Yet the role of insulators in plants remains poorly understood, in large part because of a lack of well-characterized insulators and binding factor(s). In this study, we isolated a 1.2-kb RS2-9 insulator from the Oryza sativa (rice) genome that can, when interposed between an enhancer and promoter, efficiently block the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and Nicotiana tabacum (tobacco). In the rice genome, the genes flanking RS2-9 exhibit an absence of mutual transcriptional interactions, as well as a lack of histone modification spread. We further determined that O. sativa Homeobox 1 (OSH1) bound two regions of RS2-9, as well as over 50 000 additional sites in the rice genome, the majority of which resided in intergenic regions. Mutation of one of the two OSH1-binding sites in RS2-9 impaired insulation activity by up to 60%, whereas the mutation of both binding sites virtually abolished insulator function. We also demonstrated that OSH1 binding sites were associated with 72% of the boundaries of topologically associated domains (TADs) identified in the rice genome, which is comparable to the 77% of TAD boundaries bound by the insulator CCCTC-binding factor (CTCF) in mammals. Taken together, our findings indicate that OSH1-RS2-9 acts as a true insulator in plants, and highlight a potential role for OSH1 in gene insulation and topological organization in plant genomes.

Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize.

Recent advances in the development of CRISPR-Cas genome editing technologies have made it possible to perform targeted mutagenesis and precise gene replacement in crop plants. CRISPR-Cas9 and CRISPR-Cas12a are two main types of widely used genome editing systems. However, when CRISPR-Cas12a editing machinery is expressed from a transgene, some chromosomal targets encountered low editing frequency in important crops like maize and soybean. Here, we report efficient methods to directly generate genome edited lines by delivering Cas12a-gRNA ribonucleoprotein complex (RNP) to immature maize embryos through particle bombardment in an elite maize variety. Genome edited lines were obtained at ~7% frequency without any selection during regeneration via biolistic delivery of Cas12a RNP into immature embryos. Strikingly, the gene editing rate was increased to 60% on average and up to 100% in some experiments when the Cas12a RNP was co-delivered with a PMI selectable marker gene cassette and the induced callus cultures were selected with mannose. We also show that use of higher activity Cas12a mutants resulted in improved editing efficiency in more recalcitrant target sequence. The advances described here provide useful tools for genetic improvement of maize.

Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems.

CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis. This study greatly expands the targeting scope of Cas12a for crop genome engineering.

Morphogenic Regulators and Their Application in Improving Plant Transformation.

Generation of plant lines with transgene or edited gene variants is the desired outcome of transformation technology. Conventional DNA-based plant transformation methods are the most commonly used technology but these approaches are limited to a small number of plant species with efficient transformation systems. The ideal transformation technologies are those that allow biotechnology applications across wide genetic background, especially within elite germplasm of major crop species. This chapter will briefly review key regulatory genes involved in plant morphogenesis with a focus on in vitro somatic embryogenesis and their application in improving plant transformation.

Generation of paternal haploids in wheat by genome editing of the centromeric histone CENH3.

New breeding technologies accelerate germplasm improvement and reduce the cost of goods in seed production1-3. Many such technologies could use in vivo paternal haploid induction (HI), which occurs when double fertilization precedes maternal (egg cell) genome loss. Engineering of the essential CENTROMERIC HISTONE (CENH3) gene induces paternal HI in Arabidopsis4-6. Despite conservation of CENH3 function across crops, CENH3-based HI has not been successful outside of the Arabidopsis model system7. Here we report a commercially operable paternal HI line in wheat with a ~7% HI rate, identified by screening genome-edited TaCENH3α-heteroallelic combinations. Unlike in Arabidopsis, edited alleles exhibited reduced transmission in female gametophytes, and heterozygous genotypes triggered higher HI rates than homozygous combinations. These developments might pave the way for the deployment of CENH3 HI technology in diverse crops.

A study on optimization of pat gene expression cassette for maize transformation.

Phosphinothricin acetyltransferase gene (pat) is an important selectable marker and also a key herbicide trait gene in several commercial products. In maize, the transformation frequency (TF) using pat as a selectable marker is the lowest among the commonly used marker options including epsps, pmi or ppo. Low pat transformation efficiency can become a major bottleneck in our ability to efficiently produce large numbers of events, especially for large molecular stack vectors with multiple trait gene cassettes. The root cause of the lower efficiency of pat in maize is not well understood and it is possible that the causes are multifaceted, including maize genotype, pat marker cassette, trait gene combinations and selection system. In this work we have identified a new variant of pat gene through codon optimization that consistently produced a higher transformation frequency (> 2x) than an old version of the pat gene that has codons optimized for expression in dicot plants. The level of PAT protein in all 16 constructs was also found multifold higher (up to 40 fold) over that of the controls. All of the T0 low copy transgenic plants generated from the 16 different constructs showed excellent tolerance to ammonium glufosinate herbicide spray tests at 4x and 8x recommended field application rates (1x = 595 g active ingredient (ai)/hectare of ammonium glufosinate) in the greenhouse.

One-step genome editing of elite crop germplasm during haploid induction.

Genome editing using CRISPR-Cas9 works efficiently in plant cells1, but delivery of genome-editing machinery into the vast majority of crop varieties is not possible using established methods2. We co-opted the aberrant reproductive process of haploid induction (HI)3-6 to induce edits in nascent seeds of diverse monocot and dicot species. Our method, named HI-Edit, enables direct genomic modification of commercial crop varieties. HI-Edit was tested in field and sweet corn using a native haploid-inducer line4 and extended to dicots using an engineered CENH3 HI system7. We also recovered edited wheat embryos using Cas9 delivered by maize pollen. Our data indicate that a transient hybrid state precedes uniparental chromosome elimination in maize HI. Edited haploid plants lack both the haploid-inducer parental DNA and the editing machinery. Therefore, edited plants could be used in trait testing and directly integrated into commercial variety development.

Plant DNA Repair Pathways and Their Applications in Genome Engineering.

Remarkable progress in the development of technologies for sequence-specific modification of primary DNA sequences has enabled the precise engineering of crops with novel characteristics. These programmable sequence-specific modifiers include site-directed nucleases (SDNs) and base editors (BEs). Currently, these genome editing machineries can be targeted to specific chromosomal locations to induce sequence changes. However, the sequence mutation outcomes are often greatly influenced by the type of DNA damage being generated, the status of host DNA repair machinery, and the presence and structure of DNA repair donor molecule. The outcome of sequence modification from repair of DNA double-strand breaks (DSBs) is often uncontrollable, resulting in unpredictable sequence insertions or deletions of various sizes. For base editing, the precision of intended edits is much higher, but the efficiency can vary greatly depending on the type of BE used or the activity of the endogenous DNA repair systems. This article will briefly review the possible DNA repair pathways present in the plant cells commonly used for generating edited variants for genome engineering applications. We will discuss the potential use of DNA repair mechanisms for developing and improving methodologies to enhance genome engineering efficiency and to direct DNA repair processes toward the desired outcomes.

Repurposing Macromolecule Delivery Tools for Plant Genetic Modification in the Era of Precision Genome Engineering.

Efficient delivery of macromolecules into plant cells and tissues is important for both basic research and biotechnology product applications. In transgenic research, the goal is to deliver DNA molecules into regenerable cells and stably integrate them into the genome. Over the past 40 years, many macromolecule delivery methods have been studied. To generate transgenic plants, particle bombardment and Agrobacterium-mediated transformation are the methods of choice for DNA delivery. The rapid advance of genome editing technologies has generated new requirements on large biomolecule delivery and at the same time reinvigorated the development of new transformation technologies. Many of the gene delivery options that have been studied before are now being repurposed for delivering genome editing machinery for various applications. This article reviews the major progress in the development of tools for large biomolecule delivery into plant cells in the new era of precision genome engineering.

Acetosyringone treatment duration affects large T-DNA molecule transfer to rice callus.

BACKGROUND: Large T-DNA fragment transfer has long been a problem for Agrobacterium-mediated transformation. Although vector systems, such as the BIBAC series, were successfully developed for the purpose, low transformation efficiencies were consistently observed. RESULTS: To gain insights of this problem in monocot transformation, we investigated the T-strand accumulation of various size of T-DNA in two kinds of binary vectors (one copy vs. multi-copy) upon acetosyringone (AS) induction and explored ways to improve the efficiency of the large T-DNA fragment transfer in Agrobacterium-mediated rice transformation. By performing immuno-precipitation of VirD2-T-strands and quantitative real-time PCR assays, we monitored the accumulation of the T-strands in Agrobacterium tumeficiens after AS induction. We further demonstrated that extension of AS induction time highly significantly improved large-size T-DNA transfer to rice cells. CONCLUSIONS: Our data provide valuable information of the T-strand dynamics and its impact on large T-DNA transfer in monocots, and likely dicots as well.

Advances in Agrobacterium-mediated Maize Transformation.

One of the major limitations of maize transformation is the isolation of a large number of immature embryos using the time-consuming manual extraction method. In this article, we describe a novel bulk embryo extraction method for fast isolation of a large number of embryos suitable for both biolistic- and Agrobacterium-mediated transformation. Optimal gene delivery and tissue culture conditions are also described for achieving high efficiency in Agrobacterium-mediated maize transformation using phosphomannose isomerase (PMI) as a selectable marker.

Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize.

Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments.

Maize transformation technology development for commercial event generation.

Maize is an important food and feed crop in many countries. It is also one of the most important target crops for the application of biotechnology. Currently, there are more biotech traits available on the market in maize than in any other crop. Generation of transgenic events is a crucial step in the development of biotech traits. For commercial applications, a high throughput transformation system producing a large number of high quality events in an elite genetic background is highly desirable. There has been tremendous progress in Agrobacterium-mediated maize transformation since the publication of the Ishida et al. (1996) paper and the technology has been widely adopted for transgenic event production by many labs around the world. We will review general efforts in establishing efficient maize transformation technologies useful for transgenic event production in trait research and development. The review will also discuss transformation systems used for generating commercial maize trait events currently on the market. As the number of traits is increasing steadily and two or more modes of action are used to control key pests, new tools are needed to efficiently transform vectors containing multiple trait genes. We will review general guidelines for assembling binary vectors for commercial transformation. Approaches to increase transformation efficiency and gene expression of large gene stack vectors will be discussed. Finally, recent studies of targeted genome modification and transgene insertion using different site-directed nuclease technologies will be reviewed.

Trait stacking in transgenic crops: challenges and opportunities.

In recent years, there has been a rapid increase in the planting of transgenic crops with stacked traits. Most of these products have been formed by conventional breeding, i.e. the crossing of transgenic plant (event) containing individual transgenes with other event(s) containing single or double transgenic traits. Many biotech companies are developing stacked trait products with increasing numbers of insect and herbicide tolerance genes for controlling a broad range of insect pests and weeds. There has also been an increase in development of technologies for molecular stacking of multiple traits in a single transgene locus. In this review we look at the status of stacked trait products, crop trait stacking technologies and the technical challenges we are facing. We also review recent progress in developing technology for assembling large transgene arrays in vitro (molecular stacks), their delivery to crop plants and issues they pose for transgene expression.

Targeted integration of T-DNA into the tobacco genome at double-stranded breaks: new insights on the mechanism of T-DNA integration.

Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome. We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease. Sequencing of genomic DNA/T-DNA junctions in targeted events revealed that genomic DNA at the cleavage sites was usually intact or nearly so, whereas donor T-DNA ends were often resected, sometimes extensively, as is found in random T-DNA inserts. Short filler DNAs were also present in several junctions. When an I-CeuI site was placed in the donor T-DNA, it was often cleaved by I-CeuI endonuclease, leading to precisely truncated targeted T-DNA inserts. Their structure requires that T-DNA cutting occurred before or during integration, indicating that T-DNA is at least partially double stranded before integration is complete. This method of targeting full-length T-DNA with considerable fidelity to a chosen break point in the plant genome may have experimental and practical applications. Our findings suggest that insertion at break points by nonhomologous end joining is one normal mode of entry for T-DNA into the plant genome.

Research note: Maternally-controlled ovule abortion results from cosuppression of dihydroflavonol-4-reductase or flavonoid-3',5'-hydroxylase genes in Petunia hybrida.

Transgenes designed to overexpress anthocyanin genes An6 (encoding dihydroflavonol-4-reductase) or Hf1 (encoding flavonoid-3',5'-hydroxylase) in Petunia hybrida L. produced flower colour phenotypes similar to those caused by sense cosuppression of chalcone synthase (Chs) genes. However, unlike Chs, sense cosuppression of An6 and Hf1 resulted in female infertility in transgenotes exhibiting complete phenotypic suppression of anthocyanins. Female sterility appeared to be due to embryo abortion, with discolouration of ovules first appearing about 4 d post-fertilization, followed by gradual collapse of the ovule. Pollen from cosuppressed, female-sterile transgenotes placed on wild-type stigmas produced normal seed set, indicating that sterility of cosuppressed plants was maternally controlled. We suggest an hypothesis that cosuppression of An6 and Hf1 leads to accumulation of dihydroflavonols in the seed coat, a maternal tissue, and that this accumulation inhibits embryo growth, either directly or indirectly. In this hypothesis, direct inhibition of embryo growth would require that dihydroflavonols diffuse from the seed coat into the embryo and act there, whereas indirect inhibition would require that dihydroflavonols interfere with some capacity of the seed coat to promote embryo growth.