CRISPR DNA- and RNP-Mediated Genome Editing via Nicotiana benthamiana Protoplast Transformation and Regeneration.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated system) has become the multipurpose tool to manipulate plant genome via their programmable sequence recognition, binding, and cleavage activities. Efficient plant genome modification often requires robust plant transformation. For most plant species, the CRISPR/Cas reagents are delivered into plants as plasmids by Agrobacterium-mediated T-DNA transfer or biolistic approaches. However, these methods are generally inefficient, heavily genotype dependent, and low throughput. Among the alternative plant transformation approaches, the protoplast-based transformation holds the potential to directly deliver DNA, RNA, or protein molecules into plant cells in an efficient and high-throughput manner. Here, we presented a robust and simplified protocol for protoplast-based DNA/ribonucleoprotein (RNP )-mediated genome editing in the model species Nicotiana benthamiana. Using this protocol, we have achieved the gene editing efficiency at 30-60% in protoplasts and 50-80% in regenerated calli and plants. The edited protoplasts can be readily regenerated without selection agents owing to highly efficient DNA or preassembled RNP transformation frequency. Lastly, this protocol utilized an improved culture media regime to overcome the complex media composition used in the previous studies. It offers quick turnaround time and higher throughput to facilitate the development of new genetic engineering technologies and holds the promise to combine with other genetic and genomic tools for fundamental and translational plant research.

Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants.

Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7-41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0-85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants.

Biolistic Transformation of Japonica Rice Varieties.

Genetic improvement of rice is crucial to achieve global food security as rice is an important staple crop for more than half of the global population. One of the methodologies for genetic improvement is biolistic delivery of genetic components into plant cells. In this chapter, we describe steps involved in introducing plasmid DNA carrying gene of interest into rice mature embryos using Biolistic® PDS-1000/He particle delivery system. We also provide information required for recovery of transformed plants and production of transgenic seed for next generation analysis. Using this protocol, typical 50-70 putative independent transgenic callus lines can be generated from 100 bombarded embryos. Transgenic rice plantlets can be produced within 2 months after the initiation of seed germination for transformation.

Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene.

BACKGROUND: Delivery of CRISPR reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes. Another technical advantage is that RNP delivery can bypass the need of cloning and vector construction steps. In this work we compared efficacies and types of edits for three Cas9 (WT Cas9 nuclease, HiFi Cas9 nuclease, Cas9 D10A nickase) and two Cas12a nucleases (AsCas12a and LbCas12a), using the rice phytoene desaturase (PDS) gene as a target site. FINDINGS: Delivery of two Cas9 nucleases (WT Cas9, and HiFi Cas9) and one Cas12a nuclease (LbCas12a) resulted in targeted mutagenesis of the PDS gene. LbCas12a had a higher editing efficiency than that of WT Cas9 and HiFi Cas9. Editing by Cas9 enzymes resulted in indels (1-2 bp) or larger deletions between 20-bp to 30-bp, which included the loss of the PAM site; whereas LbCas12a editing resulted in deletions ranging between 2 bp to 20 bp without the loss of the PAM site. CONCLUSIONS: In this work, when a single target site of the rice gene OsPDS was evaluated, the LbCas12a RNP complex achieved a higher targeted mutagenesis frequency than the AsCas12a or Cas9 RNPs.

High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice.

An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.

The ratio of phytosiderophores nicotianamine to deoxymugenic acid controls metal homeostasis in rice.

MAIN CONCLUSION: The ratio of nicotianamine to deoxymugenic acid controls tissue-specific metal homeostasis in rice and regulates metal delivery to the endosperm. The metal-chelating phytosiderophores nicotianamine (NA) and 2'deoxymugenic acid (DMA) are significant factors for the control of metal homeostasis in graminaceous plants. These compounds are thought to influence metal homeostasis, but their individual roles and the effect of altering the NA:DMA ratio are unknown. We purposely generated rice lines with high and low NA:DMA ratios (HND and LND lines, respectively). The HND lines accumulated more iron (Fe), zinc (Zn), manganese (Mn) and copper (Cu) in the endosperm through the mobilization of Fe, Zn and Mn from the seed husk to the endosperm. In contrast, Fe, Zn and Mn were mobilized to the husk in the LND lines, whereas Cu accumulated in the endosperm. Different groups of metals are, therefore, taken up, transported and sequestered in vegetative tissues in the HND and LND lines to achieve this metal distribution pattern in the seeds. We found that different sets of endogenous metal homeostasis genes were modulated in the HND and LND lines to achieve differences in metal homeostasis. Our findings demonstrate that the NA:DMA ratio is a key factor regulating metal homeostasis in graminaceous plants. These findings can help formulate refined strategies to improve nutrient composition and nutrient use efficiency in crop plants.

CRISPR/Cas9-mediated targeted T-DNA integration in rice.

KEY MESSAGE: Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome. Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50-80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.

Iron and Zinc in the Embryo and Endosperm of Rice (Oryza sativa L.) Seeds in Contrasting 2'-Deoxymugineic Acid/Nicotianamine Scenarios.

Iron and Zn deficiencies are worldwide nutritional disorders that can be alleviated by increasing the metal concentration of rice (Oryza sativa L.) grains via bio-fortification approaches. The overproduction of the metal chelator nicotianamine (NA) is among the most effective ones, but it is still unclear whether this is due to the enrichment in NA itself and/or the concomitant enrichment in the NA derivative 2'-deoxymugineic acid (DMA). The endosperm is the most commonly consumed portion of the rice grain and mediates the transfer of nutrients from vegetative tissues to the metal rich embryo. The impact of contrasting levels of DMA and NA on the metal distribution in the embryo and endosperm of rice seeds has been assessed using wild-type rice and six different transgenic lines overexpressing nicotianamine synthase (OsNAS1) and/or barley nicotianamine amino transferase (HvNAATb). These transgenic lines outlined three different DMA/NA scenarios: (i) in a first scenario, an enhanced NA level (via overexpression of OsNAS1) would not be fully depleted because of a limited capacity to use NA for DMA synthesis (lack of -or low- expression of HvNAATb), and results in consistent enrichments in NA, DMA, Fe and Zn in the endosperm and NA, DMA and Fe in the embryo; (ii) in a second scenario, an enhanced NA level (via overexpression of OsNAS1) would be depleted by an enhanced capacity to use NA for DMA synthesis (via expression of HvNAATb), and results in enrichments only for DMA and Fe, both in the endosperm and embryo, and (iii) in a third scenario, the lack of sufficient NA replenishment would limit DMA synthesis, in spite of the enhanced capacity to use NA for this purpose (via expression of HvNAATb), and results in decreases in NA, variable changes in DMA and moderate decreases in Fe in the embryo and endosperm. Also, quantitative LA-ICP-MS metal map images of the embryo structures show that the first and second scenarios altered local distributions of Fe, and to a lesser extent of Zn. The roles of DMA/NA levels in the transport of Fe and Zn within the embryo are thoroughly discussed.

Phytosiderophores determine thresholds for iron and zinc accumulation in biofortified rice endosperm while inhibiting the accumulation of cadmium.

Nicotianamine (NA) and 2'-deoxymugenic acid (DMA) are metal-chelating ligands that promote the accumulation of metals in rice endosperm, but it is unclear how these phytosiderophores regulate the levels of different metals and limit their accumulation. In this study, transgenic rice plants producing high levels of NA and DMA accumulated up to 4-fold more iron (Fe) and 2-fold more zinc (Zn) in the endosperm compared with wild-type plants. The distribution of Fe and Zn in vegetative tissues suggested that both metals are sequestered as a buffering mechanism to avoid overloading the seeds. The buffering mechanism involves the modulation of genes encoding metal transporters in the roots and aboveground vegetative tissues. As well as accumulating more Fe and Zn, the endosperm of the transgenic plants accumulated less cadmium (Cd), suggesting that higher levels of Fe and Zn competitively inhibit Cd accumulation. Our data show that although there is a strict upper limit for Fe (~22.5 µg g-1 dry weight) and Zn (~84 µg g-1 dry weight) accumulation in the endosperm, the careful selection of strategies to increase endosperm loading with essential minerals can also limit the accumulation of toxic metals such as Cd, thus further increasing the nutritional value of rice.

The expression of heterologous Fe (III) phytosiderophore transporter HvYS1 in rice increases Fe uptake, translocation and seed loading and excludes heavy metals by selective Fe transport.

Many metal transporters in plants are promiscuous, accommodating multiple divalent cations including some which are toxic to humans. Previous attempts to increase the iron (Fe) and zinc (Zn) content of rice endosperm by overexpressing different metal transporters have therefore led unintentionally to the accumulation of copper (Cu), manganese (Mn) and cadmium (Cd). Unlike other metal transporters, barley Yellow Stripe 1 (HvYS1) is specific for Fe. We investigated the mechanistic basis of this preference by constitutively expressing HvYS1 in rice under the control of the maize ubiquitin1 promoter and comparing the mobilization and loading of different metals. Plants expressing HvYS1 showed modest increases in Fe uptake, root-to-shoot translocation, seed accumulation and endosperm loading, but without any change in the uptake and root-to-shoot translocation of Zn, Mn or Cu, confirming the selective transport of Fe. The concentrations of Zn and Mn in the endosperm did not differ significantly between the wild-type and HvYS1 lines, but the transgenic endosperm contained significantly lower concentrations of Cu. Furthermore, the transgenic lines showed a significantly reduced Cd uptake, root-to-shoot translocation and accumulation in the seeds. The underlying mechanism of metal uptake and translocation reflects the down-regulation of promiscuous endogenous metal transporters revealing an internal feedback mechanism that limits seed loading with Fe. This promotes the preferential mobilization and loading of Fe, therefore displacing Cu and Cd in the seed.

Engineering metabolic pathways in plants by multigene transformation.

Metabolic engineering in plants can be used to increase the abundance of specific valuable metabolites, but single-point interventions generally do not improve the yields of target metabolites unless that product is immediately downstream of the intervention point and there is a plentiful supply of precursors. In many cases, an intervention is necessary at an early bottleneck, sometimes the first committed step in the pathway, but is often only successful in shifting the bottleneck downstream, sometimes also causing the accumulation of an undesirable metabolic intermediate. Occasionally it has been possible to induce multiple genes in a pathway by controlling the expression of a key regulator, such as a transcription factor, but this strategy is only possible if such master regulators exist and can be identified. A more robust approach is the simultaneous expression of multiple genes in the pathway, preferably representing every critical enzymatic step, therefore removing all bottlenecks and ensuring completely unrestricted metabolic flux. This approach requires the transfer of multiple enzyme-encoding genes to the recipient plant, which is achieved most efficiently if all genes are transferred at the same time. Here we review the state of the art in multigene transformation as applied to metabolic engineering in plants, highlighting some of the most significant recent advances in the field.

Paradoxical EU agricultural policies on genetically engineered crops.

European Union (EU) agricultural policy has been developed in the pursuit of laudable goals such as a competitive economy and regulatory harmony across the union. However, what has emerged is a fragmented, contradictory, and unworkable legislative framework that threatens economic disaster. In this review, we present case studies highlighting differences in the regulations applied to foods grown in EU countries and identical imported products, which show that the EU is undermining its own competitiveness in the agricultural sector, damaging both the EU and its humanitarian activities in the developing world. We recommend the adoption of rational, science-based principles for the harmonization of agricultural policies to prevent economic decline and lower standards of living across the continent.

Can the world afford to ignore biotechnology solutions that address food insecurity?

Genetically engineered (GE) crops can be used as part of a combined strategy to address food insecurity, which is defined as a lack of sustainable access to safe and nutritious food. In this article, we discuss the causes and consequences of food insecurity in the developing world, and the indirect economic impact on industrialized countries. We dissect the healthcare costs and lost productivity caused by food insecurity, and evaluate the relative merits of different intervention programs including supplementation, fortification and the deployment of GE crops with higher yields and enhanced nutritional properties. We provide clear evidence for the numerous potential benefits of GE crops, particularly for small-scale and subsistence farmers. GE crops with enhanced yields and nutritional properties constitute a vital component of any comprehensive strategy to tackle poverty, hunger and malnutrition in developing countries and thus reduce the global negative economic effects of food insecurity.

Biofortification of plants with altered antioxidant content and composition: genetic engineering strategies.

Antioxidants are protective molecules that neutralize reactive oxygen species and prevent oxidative damage to cellular components such as membranes, proteins and nucleic acids, therefore reducing the rate of cell death and hence the effects of ageing and ageing-related diseases. The fortification of food with antioxidants represents an overlap between two diverse environments, namely fortification of staple foods with essential nutrients that happen to have antioxidant properties (e.g. vitamins C and E) and the fortification of luxury foods with health-promoting but non-essential antioxidants such as flavonoids as part of the nutraceuticals/functional foods industry. Although processed foods can be artificially fortified with vitamins, minerals and nutraceuticals, a more sustainable approach is to introduce the traits for such health-promoting compounds at source, an approach known as biofortification. Regardless of the target compound, the same challenges arise when considering the biofortification of plants with antioxidants, that is the need to modulate endogenous metabolic pathways to increase the production of specific antioxidants without affecting plant growth and development and without collateral effects on other metabolic pathways. These challenges become even more intricate as we move from the engineering of individual pathways to several pathways simultaneously. In this review, we consider the state of the art in antioxidant biofortification and discuss the challenges that remain to be overcome in the development of nutritionally complete and health-promoting functional foods.

The contribution of transgenic plants to better health through improved nutrition: opportunities and constraints.

Malnutrition is a prevalent and entrenched global socioeconomic challenge that reflects the combined impact of poverty, poor access to food, inefficient food distribution infrastructure, and an over-reliance on subsistence mono-agriculture. The dependence on staple cereals lacking many essential nutrients means that malnutrition is endemic in developing countries. Most individuals lack diverse diets and are therefore exposed to nutrient deficiencies. Plant biotechnology could play a major role in combating malnutrition through the engineering of nutritionally enhanced crops. In this article, we discuss different approaches that can enhance the nutritional content of staple crops by genetic engineering (GE) as well as the functionality and safety assessments required before nutritionally enhanced GE crops can be deployed in the field. We also consider major constraints that hinder the adoption of GE technology at different levels and suggest policies that could be adopted to accelerate the deployment of nutritionally enhanced GE crops within a multicomponent strategy to combat malnutrition.

Bacillus thuringiensis: a century of research, development and commercial applications.

Bacillus thuringiensis (Bt) is a soil bacterium that forms spores during the stationary phase of its growth cycle. The spores contain crystals, predominantly comprising one or more Cry and/or Cyt proteins (also known as δ-endotoxins) that have potent and specific insecticidal activity. Different strains of Bt produce different types of toxin, each of which affects a narrow taxonomic group of insects. Therefore, Bt toxins have been used as topical pesticides to protect crops, and more recently the proteins have been expressed in transgenic plants to confer inherent pest resistance. Bt transgenic crops have been overwhelmingly successful and beneficial, leading to higher yields and reducing the use of chemical pesticides and fossil fuels. However, their deployment has attracted some criticism particularly with regard to the potential evolution of pest-resistant insect strains. Here, we review recent progress in the development of Bt technology and the countermeasures that have been introduced to prevent the evolution of resistant insect populations.

The potential impact of plant biotechnology on the Millennium Development Goals.

The eight Millennium Development Goals (MDGs) are international development targets for the year 2015 that aim to achieve relative improvements in the standards of health, socioeconomic status and education in the world's poorest countries. Many of the challenges addressed by the MDGs reflect the direct or indirect consequences of subsistence agriculture in the developing world, and hence, plant biotechnology has an important role to play in helping to achieve MDG targets. In this opinion article, we discuss each of the MDGs in turn, provide examples to show how plant biotechnology may be able to accelerate progress towards the stated MDG objectives, and offer our opinion on the likelihood of such technology being implemented. In combination with other strategies, plant biotechnology can make a contribution towards sustainable development in the future although the extent to which progress can be made in today's political climate depends on how we deal with current barriers to adoption.