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Light absorbed by chlorophylls of photosystem II and I drives oxygenic photosynthesis. Light-harvesting complexes increase the absorption cross-section of these photosystems. Furthermore, these complexes play a central role in photoprotection by dissipating the excess of absorbed light energy in an inducible and regulated fashion. In higher plants, the main light-harvesting complex is the trimeric LHCII.In this work, we used CRISPR/Cas9 to knockout the five genes encoding LHCB1, which is the major component of the trimeric LHCII. In absence of LHCB1 the accumulation of the other LHCII isoforms was only slightly increased, thereby resulting in chlorophyll loss leading to a pale green phenotype and growth delay. Photosystem II absorption cross-section was smaller while photosystem I absorption cross-section was unaffected. This altered the chlorophyll repartition between the two photosystems, favoring photosystem I excitation. The equilibrium of the photosynthetic electron transport was partially maintained by a lower photosystem I over photosystem II reaction center ratio and by the dephosphorylation of LHCII and photosystem II. Loss of LHCB1 altered the thylakoid structure, with less membrane layers per grana stack and reduced grana width. Stable LHCB1 knock out lines allow characterizing the role of this protein in light harvesting and acclimation and pave the way for future in vivo mutational analyses of LHCII.

Breeding of stress-tolerant plants is able to improve crop yield under stress conditions, whereas CRISPR/Cas9 genome editing has been shown to be an efficient way for molecular breeding to improve agronomic traits including stress tolerance in crops. However, genes can be targeted for genome editing to enhance crop abiotic stress tolerance remained largely unidentified. We have previously identified abscisic acid (ABA)-induced transcription repressors (AITRs) as a novel family of transcription factors that are involved in the regulation of ABA signaling, and we found that knockout of the entire family of AITR genes in Arabidopsis enhanced drought and salinity tolerance without fitness costs. Considering that AITRs are conserved in angiosperms, AITRs in crops may be targeted for genome editing to improve abiotic stress tolerance. We report here that mutation of GmAITR genes by CRISPR/Cas9 genome editing leads to enhanced salinity tolerance in soybean. By using quantitative RT-PCR analysis, we found that the expression levels of GmAITRs were increased in response to ABA and salt treatments. Transfection assays in soybean protoplasts show that GmAITRs are nucleus proteins, and have transcriptional repression activities. By using CRISPR/Cas9 to target the six GmAITRs simultaneously, we successfully generated Cas9-free gmaitr36 double and gmaitr23456 quintuple mutants. We found that ABA sensitivity in these mutants was increased. Consistent with this, ABA responses of some ABA signaling key regulator genes in the gmaitr mutants were altered. In both seed germination and seedling growth assays, the gmaitr mutants showed enhanced salt tolerance. Most importantly, enhanced salinity tolerance in the mutant plants was also observed in the field experiments. These results suggest that mutation of GmAITR genes by CRISPR/Cas9 is an efficient way to improve salinity tolerance in soybean.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful tool for the precise editing of plant genomes for crop improvement. Rapid in vitro methods for the determination of guide RNA (gRNA) cleavage efficiency and an efficient DNA delivery system is essential for gene editing. However, we lack an efficient gene-editing system for palm species. In this study, we described the development of a transient oil palm protoplast assay to rapidly evaluate the cleavage efficiency of CRISPR/Cas9 mutagenesis and the generation of stable transformed oil palms using biolistic particle bombardment in immature embryos. Using the phytoene desaturase (EgPDS) gene, we found cleavage frequency of up to 25.49% in electro-transfected protoplast, which enables the production of transgenic oil palm shoots exhibiting chimeric albino phenotypes as a result of DNA insertions, deletions (InDels), and nucleotide substitutions, with a mutation efficiency of 62.5–83.33%. We further validated the mutagenesis efficiency and specificity of the CRISPR/Cas9 system in oil palm by targeting the brassinosteroid-insensitive 1 (EgBRI1) gene, which resulted in nucleotide substitutions in EgBRI1 with premature necrosis phenotype in oil palm transgenic shoots and stunted phenotype resulting from DNA InDels. Taken together, our results showed that effective and efficient editing of genes using the CRISPR/Cas9 system can be achieved in oil palm by optimizing the selection of efficient gRNA and DNA delivery methods. This newly designed strategy will enable new routes for the genetic improvement in oil palm and related species.

Conifers are the world's major source of timber and pulpwood and have great economic and ecological value. Currently, little research on the application of CRISPR/Cas9, the commonly used genome-editing tool in angiosperms, has been reported in coniferous species. An efficient CRISPR/Cas9 system based on somatic embryogenesis (SEis) suitable for conifers could benefit both fundamental and applied research in these species. In this study, the SpCas9 gene was optimized based on codon bias in white spruce, and a spruce U6 promoter was cloned and function-validated for use in a conifer specific CRISPR/Cas9 toolbox, i.e., PgCas9/PaU6. With this toolbox, a genome-editing vector was constructed to target the DXS1 gene of white spruce. By Agrobacterium-mediated transformation, the genome-editing vector was then transferred into embryogenic tissue of white spruce. Three resistant embryogenic tissues were obtained and used for regenerating plants via SEis. Albino somatic embryo (SE) plants with mutations in DXS1 were obtained in all of the three events, and the ratios of the homozygous and biallelic mutants in the 18 albino mutants detected were 22.2% in both cases. Green plants with mutations in DXS1 were also produced, and the ratios of the DXS1 mutants to the total green plants were 7.9, 28, and 13.5%, respectively, among the three events. Since 22.7% of the total 44 mutants were edited at both of the target sites 1 and 2, the CRISPR/Cas9 toolbox in this research could be used for multi-sites genome editing. More than 2,000 SE plants were regenerated in vitro after genome editing, and part of them showed differences in plant development. Both chimerism and mosaicism were found in the SE plants of white spruce after genome editing with the CRISPR/Cas9 toolbox. The conifer-specific CRISPR/Cas9 system developed in this research could be valuable in gene function research and trait improvement.

Multiplex genome-editing (MGE) technologies are recently developed versatile bioengineering tools for modifying two or more specific DNA loci in a genome with high precision. These genome-editing tools have greatly increased the feasibility of introducing desired changes at multiple nucleotide levels into a target genome. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) [CRISPR/Cas] system-based MGE tools allow the simultaneous generation of direct mutations precisely at multiple loci in a gene or multiple genes. MGE is enhancing the field of plant molecular biology and providing capabilities for revolutionizing modern crop-breeding methods as it was virtually impossible to edit genomes so precisely at the single base-pair level with prior genome-editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Recently, researchers have not only started using MGE tools to advance genome-editing applications in certain plant science fields but also have attempted to decipher and answer basic questions related to plant biology. In this review, we discuss the current progress that has been made toward the development and utilization of MGE tools with an emphasis on the improvements in plant biology after the discovery of CRISPR/Cas9. Furthermore, the most recent advancements involving CRISPR/Cas applications for editing multiple loci or genes are described. Finally, insights into the strengths and importance of MGE technology in advancing crop-improvement programs are presented.

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase (pds), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological applications

Numerous important pharmaceuticals and nutraceuticals originate from plant specialized metabolites, most of which are synthesized via complex biosynthetic pathways. The elucidation of these pathways is critical for the applicable uses of these compounds. Although the rapid progress of the omics technology has revolutionized the identification of candidate genes involved in these pathways, the functional characterization of these genes remains a major bottleneck. Baker’s yeast (Saccharomyces cerevisiae) has been used as a microbial platform for characterizing newly discovered metabolic genes in plant specialized metabolism. Using yeast for the investigation of numerous plant enzymes is a streamlined process because of yeast’s efficient transformation, limited endogenous specialized metabolism, partially sharing its primary metabolism with plants, and its capability of post-translational modification. Despite these advantages, reconstructing complex plant biosynthetic pathways in yeast can be time intensive. Since its discovery, CRISPR/Cas9 has greatly stimulated metabolic engineering in yeast. Yeast is a popular system for genome editing due to its efficient homology-directed repair mechanism, which allows precise integration of heterologous genes into its genome. One practical use of CRISPR/Cas9 in yeast is multiplex genome editing aimed at reconstructing complex metabolic pathways. This system has the capability of integrating multiple genes of interest in a single transformation, simplifying the reconstruction of complex pathways. As plant specialized metabolites usually have complex multigene biosynthetic pathways, the multiplex CRISPR/Cas9 system in yeast is suited well for functional genomics research in plant specialized metabolism. Here, we review the most advanced methods to achieve efficient multiplex CRISPR/Cas9 editing in yeast. We will also discuss how this powerful tool has been applied to benefit the study of plant specialized metabolism.

Climate change is a threat to global food security due to the reduction of crop productivity around the globe. Food security is a matter of concern for stakeholders and policymakers as the global population is predicted to bypass 10 billion in the coming years. Crop improvement via modern breeding techniques along with efficient agronomic practices innovations in microbiome applications, and exploiting the natural variations in underutilized crops is an excellent way forward to fulfill future food requirements. In this review, we describe the next-generation breeding tools that can be used to increase crop production by developing climate-resilient superior genotypes to cope with the future challenges of global food security. Recent innovations in genomic-assisted breeding (GAB) strategies allow the construction of highly annotated crop pan-genomes to give a snapshot of the full landscape of genetic diversity (GD) and recapture the lost gene repertoire of a species. Pan-genomes provide new platforms to exploit these unique genes or genetic variation for optimizing breeding programs. The advent of next-generation clustered regularly interspaced short palindromic repeat/CRISPR-associated (CRISPR/Cas) systems, such as prime editing, base editing, and de nova domestication, has institutionalized the idea that genome editing is revamped for crop improvement. Also, the availability of versatile Cas orthologs, including Cas9, Cas12, Cas13, and Cas14, improved the editing efficiency. Now, the CRISPR/Cas systems have numerous applications in crop research and successfully edit the major crop to develop resistance against abiotic and biotic stress. By adopting high-throughput phenotyping approaches and big data analytics tools like artificial intelligence (AI) and machine learning (ML), agriculture is heading toward automation or digitalization. The integration of speed breeding with genomic and phenomic tools can allow rapid gene identifications and ultimately accelerate crop improvement programs. In addition, the integration of next-generation multidisciplinary breeding platforms can open exciting avenues to develop climate-ready crops toward global food security.

[EN] CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, the design and assembly of multiplex constructs comprising tandemly arrayed guide RNAs (gRNAs) requires scarless cloning and is still troublesome due to the presence of repetitive sequences, thus hampering a more widespread use. Here we present a comprehensive extension of the software-assisted cloning platform GoldenBraid (GB), in which, on top of its multigene cloning software, we integrate new tools for the Type IIS-based easy and rapid assembly of up to six tandemly-arrayed gRNAs with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the crRNA unspaced approaches, respectively. As stress tests for the new tools, we assembled and used for Agrobacterium-mediated stable transformation a 17 Cas9-gRNAs construct targeting a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in Nicotiana tabacum. The 14 selected genes are targets of miR156, thus potentially playing an important role in juvenile-to-adult and vegetative-to-reproductive phase transitions. With the 17 gRNAs construct we generated a collection of Cas9-free SPL edited T-1 plants harboring up to 9 biallelic mutations and showing leaf juvenility and more branching. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR/Cas activators and repressors using single and multiplexing gRNAs was validated using a Luciferase reporter with the Solanum lycopersicum Mtb promoter or the Agrobacterium tumefaciens nopaline synthase promoter in transient expression in Nicotiana benthamiana. With the incorporation of the new web-based tools and the accompanying collection of DNA parts, the GB4.0 genome edition turns an all-in-one open platform for plant genome engineering., This work had been funded by EU Horizon 2020 Project Newcotiana Grant 760331 and PID2019-108203RB-100 Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness. MV-V was recipient of aGeneralitat Valenciana and Fondo Social Europeo post-doctoral grant. JB-O and SS were recipients of FPI fellowships. CP was recipient of a Santiago Grisolia fellowship (Generalitat Valenciana).

Gene editing technology in woody plants has great potential for understanding gene function, and altering traits affecting economically and ecologically important traits. Gene editing applications in woody species require a high genome editing efficiency due to the difficulty during transformation and complexities resulting from gene redundancy. In this study, we used poplar 84K (Populus alba × P. glandulosa), which is a model hybrid for studying wood formation and growth. We developed a new CRISPR/Cas9 system to edit multiple genes simultaneously. Using this system, we successfully knocked out multiple targets of the PHYTOENE DESATURASE 8 in poplar. We found the mutation rate of our CRISPR/Cas9 system is higher (67.5%) than existing reports in woody trees. We further improved the mutation rate up to 75% at editing sites through the usage of the mannopine synthase (MAS) promoter to drive Cas9. The MAS-CRISPR/Cas9 is an improved genome-editing tool for woody plants with a higher efficiency and a higher mutation rate than currently available technologies.

Crop production has been substantially reduced by devastating fungal and oomycete pathogens, and these pathogens continue to threaten global food security. Although chemical and cultural controls have been used for crop protection, these involve continuous costs and time and fungicide resistance among plant pathogens has been increasingly reported. The most efficient way to protect crops from plant pathogens is cultivation of disease-resistant cultivars. However, traditional breeding approaches are laborious and time intensive. Recently, the CRISPR/Cas9 system has been utilized to enhance disease resistance among different crops such as rice, cacao, wheat, tomato, and grape. This system allows for precise genome editing of various organisms via RNA-guided DNA endonuclease activity. Beyond genome editing in crops, editing the genomes of fungal and oomycete pathogens can also provide new strategies for plant disease management. This review focuses on the recent studies of plant disease resistance against fungal and oomycete pathogens using the CRISPR/Cas9 system. For long-term plant disease management, the targeting of multiple plant disease resistance mechanisms with CRISPR/Cas9 and insights gained by probing fungal and oomycete genomes with this system will be powerful approaches.

The year 2020 marks a decade since the first gene-edited plants were generated using homing endonucleases and zinc finger nucleases. The advent of CRISPR/Cas9 for gene-editing in 2012 was a major science breakthrough that revolutionized both basic and applied research in various organisms including plants and consequently honored with “The Nobel Prize in Chemistry, 2020.” CRISPR technology is a rapidly evolving field and multiple CRISPR-Cas derived reagents collectively offer a wide range of applications for gene-editing and beyond. While most of these technological advances are successfully adopted in plants to advance functional genomics research and development of innovative crops, others await optimization. One of the biggest bottlenecks in plant gene-editing has been the delivery of gene-editing reagents, since genetic transformation methods are only established in a limited number of species. Recently, alternative methods of delivering CRISPR reagents to plants are being explored. This review mainly focuses on the most recent advances in plant gene-editing including (1) the current Cas effectors and Cas variants with a wide target range, reduced size and increased specificity along with tissue specific genome editing tool kit (2) cytosine, adenine, and glycosylase base editors that can precisely install all possible transition and transversion mutations in target sites (3) prime editing that can directly copy the desired edit into target DNA by search and replace method and (4) CRISPR delivery mechanisms for plant gene-editing that bypass tissue culture and regeneration procedures including de novo meristem induction, delivery using viral vectors and prospects of nanotechnology-based approaches.

The application of CRISPR/Cas9 technologies has transformed our ability to target and edit designated regions of a genome. It’s broad adaptability to any organism has led to countless advancements in our understanding of many biological processes. Many current tools are designed for simple plant systems such as diploid species, however, efficient deployment in crop species requires a greater efficiency of editing as these often contain polyploid genomes. Here, we examined the role of temperature to understand if CRISPR/Cas9 editing efficiency can be improved in wheat. The recent finding that plant growth under higher temperatures could increase mutation rates was tested with Cas9 expressed from two different promoters in wheat. Increasing the temperature of the tissue culture or of the seed germination and early growth phase increases the frequency of mutation in wheat when the Cas9 enzyme is driven by the ZmUbi promoter but not OsActin. In contrast, Cas9 expression driven by the OsActin promoter did not increase the mutations detected in either transformed lines or during the transformation process itself. These results demonstrate that CRISPR/Cas9 editing efficiency can be significantly increased in a polyploid cereal species with a simple change in growth conditions to facilitate increased mutations for the creation of homozygous or null knock-outs.

For the past 5 years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has appeared in the molecular biology research spotlight. As a game-changing player in genome editing, CRISPR/Cas9 technology has revolutionized animal research, including medical research and human gene therapy as well as plant science research, particularly for crop improvement. One of the most common applications of CRISPR/Cas9 is to generate genetic knock-out mutants. Recently, several multiplex genome editing approaches utilizing CRISPR/Cas9 were developed and applied in various aspects of plant research. Here we summarize these approaches as they relate to plants, particularly with respect to understanding the biosynthesis and function of the plant cell wall. The plant cell wall is a polysaccharide-rich cell structure that is vital to plant cell formation, growth, and development. Humans are heavily dependent on the byproducts of the plant cell wall such as shelter, food, clothes, and fuel. Genes involved in the assembly of the plant cell wall are often highly redundant. To identify these redundant genes, higher-order knock-out mutants need to be generated, which is conventionally done by genetic crossing. Compared with genetic crossing, CRISPR/Cas9 multi-gene targeting can greatly shorten the process of higher-order mutant generation and screening, which is especially useful to characterize cell wall related genes in plant species that require longer growth time. Moreover, CRISPR/Cas9 makes it possible to knock out genes when null T-DNA mutants are not available or are genetically linked. Because of these advantages, CRISPR/Cas9 is becoming an ideal and indispensable tool to perform functional studies in plant cell wall research. In this review, we provide perspectives on how to design CRISPR/Cas9 to achieve efficient gene editing and multi-gene targeting in plants. We also discuss the recent development of the virus-based CRISPR/Cas9 system and the application of CRISPR/Cas9 to knock in genes. Lastly, we summarized current progress on using CRISPR/Cas9 for the characterization of plant cell wall-related genes.

Biotic stresses caused by microbial pathogens impair crop yield and quality if not restricted by expensive and often ecologically problematic pesticides. For a sustainable agriculture of tomorrow, breeding or engineering of pathogen-resistant crop varieties is therefore a major cornerstone. Maize is one of the four most important cereal crops in the world. The biotrophic fungal pathogen Ustilago maydis causes galls on all aerial parts of the maize plant. Biotrophic pathogens like U. maydis co-evolved with their host plant and depend during their life cycle on successful manipulation of the host’s cellular machinery. Therefore, removing or altering plant susceptibility genes is an effective and usually durable way to obtain resistance in plants. Transcriptional time course experiments in U. maydis-infected maize revealed numerous maize genes being upregulated upon establishment of biotrophy. Among these genes is the maize LIPOXYGENASE 3 (LOX3) previously shown to be a susceptibility factor for other fungal genera as well. Aiming to engineer durable resistance in maize against U. maydis and possibly other pathogens, we took a Cas endonuclease technology approach to generate loss of function mutations in LOX3. lox3 maize mutant plants react with an enhanced PAMP-triggered ROS burst implicating an enhanced defense response. Based on visual assessment of disease symptoms and quantification of relative fungal biomass, homozygous lox3 mutant plants exposed to U. maydis show significantly decreased susceptibility. U. maydis infection assays using a transposon mutant lox3 maize line further substantiated that LOX3 is a susceptibility factor for this important maize pathogen.

Soybean is grown worldwide for oil and protein source as food, feed and industrial raw material for biofuel. Steady increase in soybean production in the past century mainly attributes to genetic mediation including hybridization, mutagenesis and transgenesis. However, genetic resource limitation and intricate social issues in use of transgenic technology impede soybean improvement to meet rapid increases in global demand for soybean products. New approaches in genomics and development of site-specific nucleases (SSNs) based genome editing technologies have expanded soybean genetic variations in its germplasm and have potential to make precise modification of genes controlling the important agronomic traits in an elite background. ZFNs, TALENS and CRISPR/Cas9 have been adapted in soybean improvement for targeted deletions, additions, replacements and corrections in the genome. The availability of reference genome assembly and genomic resources increases feasibility in using current genome editing technologies and their new development. This review summarizes the status of genome editing in soybean improvement and future directions in this field.

The “green revolution” gene gibberellin oxidase contributes to the semidwarf phenotype, improving product and lodging resistance. Dissecting the function of GA biosynthetic genes would be helpful for dwarf maize breeding. In this study, we edited the maize GA20ox3 gene and generated semidwarf maize plants using CRISPR/Cas9 technology. Application of exogenous gibberellin can recover the dwarf phenotype, indicating that the mutants are gibberellin deficient. The contents of GA12 and GA53 were elevated in the mutants due to the disruption of GA20 oxidase, whereas the contents of other GA precursors (GA15, GA24, GA9, GA44, and GA20) were decreased in the mutants, and the accumulation of bioactive GA1 and GA4 was also decreased, contributing to the semidwarf phenotype. Transgene-free dwarf maize was selected from T2-generation plants and might be useful for maize breeding in the future.

The β-ketoacyl-[acyl carrier protein] synthase 1 (KASI) gene has been shown in model plant systems to be critical for the conversion of sucrose to oil. A previous study characterized the morphological and seed composition phenotypes associated with a reciprocal chromosomal translocation that disrupted one of the KASI genes in soybean. The principle findings of this work included a wrinkled seed phenotype, an increase in seed sucrose, a decrease in seed oil, and a low frequency of transmission of the translocation. However, it remained unclear which, if any, of these phenotypes were directly caused by the loss of KASI gene function, as opposed to the chromosomal translocation or other associated factors. In this study, CRISPR/Cas9 mutagenesis was used to generate multiple knockout alleles for this gene, and also one in-frame allele. These soybean plants were evaluated for morphology, seed composition traits, and genetic transmission. Our results indicate that the CRISPR/Cas9 mutants exhibited the same phenotypes as the chromosomal translocation mutant, validating that the observed phenotypes are caused by the loss of gene function. Furthermore, the plants harboring homozygous in-frame mutations exhibited similar phenotypes compared to the plants harboring homozygous knockout mutations. This result indicates that the amino acids lost in the in-frame mutant are essential for proper gene function. In-frame edits for this gene may need to target less essential and/or evolutionarily conserved domains in order to generate novel seed composition phenotypes.

CRISPR/Cas9 gene editing technology has taken the scientific community by storm since its development in 2012. First discovered in 1987, CRISPR/Cas systems act as an adaptive immune response in archaea and bacteria that defends against invading bacteriophages and plasmids. CRISPR/Cas9 gene editing technology modifies this immune response to function in eukaryotic cells as a highly specific, RNA-guided complex that can edit almost any genetic target. This technology has applications in all biological fields, including plant pathology. However, examples of its use in forest pathology are essentially nonexistent. The aim of this review is to give researchers a deeper understanding of the native CRISPR/Cas systems and how they were adapted into the CRISPR/Cas9 technology used today in plant pathology – this information is crucial for researchers aiming to use this technology in the pathosystems they study. We review the current applications of CRISPR/Cas9 in plant pathology and propose future directions for research in forest pathosystems where this technology is currently underutilized.

Alfalfa (Medicago sativa) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the Medicago sativa stay-green (MsSGR) gene. The replacement of CaMV35S promoter by the Arabidopsis ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean.

The genetic engineering method CRISPR has been touted as an efficient, inexpensive, easily used, and targeted genetic modification technology that is widely suggested as having the potential to solve many of the problems facing agriculture now and in the future. Like all new technologies, however, it is not without challenges. One of the most difficult challenges to anticipate and detect is gene targets that are inaccessible due to the chromatin state at their specific location. There is currently no way to predict this during the process of designing a sgRNA target, and the only way to detect this issue before spending time and resources on full transformations is to test the cleavage ability of the sgRNA in vivo. In wheat, this is possible using protoplast isolation and PEG transformation with Cas9 ribonucleoprotein complexes. Therefore, we have developed a streamlined protocol for testing the accessibility of sgRNA targets in wheat. The first steps involve digesting wheat leaf tissue in an enzymatic solution and then isolating viable protoplasts using filters and a sucrose gradient. The protoplasts are then transformed using Cas9 ribonucleoprotein complexes via PEG-mediated transformation. DNA is isolated from the CRISPR-Cas-edited protoplasts and PCR is performed to amplify the gene target region. The PCR product is then used to assess the editing efficiency of the chosen sgRNA using Sanger sequencing. This simplified protocol for the isolation and transformation of wheat protoplast cells using Cas9 ribonucleoprotein complexes streamlines CRISPR transformation projects by allowing for a fast and easy test of sgRNA accessibility in vivo.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.

Genome editing technology represented by CRISPR-Cas9 had been widely used in many biological fields such as gene function analysis, gene therapy, and crop improvement. However, in the face of the complexity of the eukaryotic genome, the CRISPR-Cas9 genome editing tools have shown an unstable editing efficiency with large variability at different target sites. It was important to further improve the editing efficiency of the CRISPR-Cas9 system among the whole genome. In this study, based on the previous single transcription unit genome editing system (STU-SpCas9), using the ubiquitin-associated domain (UBA) to enhance the stability of Cas9 protein, we constructed three Cas9-UBA fusion systems (SpCas9-SD01, SpCas9-SD02, and SpCas9-SD03). Four different target sites of rice OsPDS, OsDEP1 and OsROC5 genes were chosen to evaluate the genome editing efficiency in rice protoplasts and stable transformed rice plants. The results showed that the fusion of UBA domains did not affect the cleavage mode of Cas9 protein, and effectively increase the editing efficiency of STU-SpCas9 at the target sites. This new CRISPR-Cas9-UBA system provided a new strategy and tool for improving the genome editing efficiency of CRISPR-Cas9 in plants.

"Easy-to-shatter" trait is a major cause of rice crop yield losses, emphasizing the economic value of developing elite rice cultivars with reduced seed shattering capable of achieving higher yields. In the present study, we describe the development of new indica rice lines that exhibit lower rates of seed shattering following the targeted CRISPR/Cas9-mediated editing of the qSH1 gene. We were able to identify qSH1 mutant T0 transgenic plants, with transgene-free homozygous mutants being obtained via segregation in the T1 generation. We then utilized two T2 transgene-free homozygous lines in order to assess the degree of seed shattering and major agronomic traits of these mutant lines and of wild-type rice plants (HR1128-WT). This approach revealed that qsh1 homozygous mutant lines exhibited significantly reduced seed shattering relative to HR1128-WT without any significant changes in other analyzed agronomic traits. We then used these mutant lines to develop new promising hybrid rice lines with intermediate seed shattering. Overall our results reveal that combining targeted gene editing via CRISPR/Cas9 with heterosis utilization approach can allow for the efficient development of novel promising hybrid rice cultivars that exhibit a intermediate of seed shattering, thereby ensuring better stability and improved rice yields.

Although the CRISPR/Cas9 system has been widely used for crop breeding, its application for the genetic improvement of trees has been limited, partly because of the outcrossing nature and substantial genomic heterozygosity of trees. Shanxin yang (Populus davidiana × P. bolleana), is a commercially important poplar clone that is widely grown in northern China. An established transformation protocol for this interspecific hybrid enables researchers to simultaneously investigate the efficiency and specificity of the CRISPR/Cas9-mediated manipulation of a highly heterozygous genome. Using the phytoene desaturase gene (PDS) as an example, we revealed that the CRISPR/Cas9 system could efficiently edit the Shanxin yang genome. Two sgRNAs were designed and incorporated into a single binary vector containing the Cas9 expression cassette. Among 62 independent transgenic lines, 85.5% exhibited an exclusively albino phenotype, revealing the total loss of PDS function. The Illumina sequencing results confirmed the targeted mutation of PdbPDS homologs induced by CRISPR/Cas9, and small insertions/deletions were the most common mutations. Biallelic and homozygous knockout mutations were detected at both target sites of the T0 transformants. Off-target activity was detected for sgRNA2 with a frequency of 3.2%. Additionally, the SNP interference of targeting specificity was assessed based on the sequence variation among PdbPDS homologs. A single mismatch at 19- or 10-bp from the PAM was tolerated by the CRISPR/Cas9 system. Therefore, multiple homologous genes were simultaneously edited despite the presence of a mismatch between the sgRNA and the target site. The establishment of a viable CRISPR/Cas9-based strategy for editing the Shanxin yang genome will not only accelerate the breeding process, but may also be relevant for other economically or scientifically important non-model plants species.

Cpf1, an endonuclease of the class 2 CRISPR family, fills the gaps that were previously faced in the world of genome engineering tools, which include the TALEN, ZFN, and CRISPR/Cas9. Other simultaneously discovered nucleases were not able to carry out re-engineering at the same region due to the loss of a target site after first-time engineering. Cpf1 acts as a dual nuclease, functioning as an endoribonuclease to process crRNA and endodeoxyribonuclease to cleave target sequences and generate double-stranded breaks. Additionally, Cpf1 allows for multiplexed genome editing, as a single crRNA array transcript can target multiple loci in the genome. The CRISPR/Cpf1 system enables gene deletion, insertion, base editing, and locus tagging in monocot as well as in dicot plants with fewer off-target effects. This tool has been efficiently demonstrated into tobacco, rice, soybean, wheat, etc. This review covers the development and applications of Cpf1 mediated genome editing technology in plants.

The CRISPR/Cas9 system is a powerful tool for targeted gene editing in many organisms including plants. However, most of the reported uses of CRISPR/Cas9 in plants have focused on modifying one or a few genes, and thus the overall specificity, types of mutations, and heritability of gene alterations remain unclear. Here we describe the molecular characterization of 361 T0 transgenic tomato plants that were generated using CRISPR/Cas9 to induce mutations in 63 immunity-associated genes. Among the T0 transformed plants, 245 carried mutations (68%), with 20% of those plants being homozygous for the mutation, 30% being heterozygous, 32% having two different mutations (biallelic) and 18% having multiple mutations (chimeric). The mutations were predominantly short insertions or deletions, with 87% of the affected sequences being smaller than 10 bp. The majority of 1 bp insertions were A (50%) or T (29%). The mutations from the T0 generation were stably transmitted to later generations, although new mutations were detected in some T1 plants. No mutations were detected in 18 potential off-target sites among 144 plants. Our study provides a broad and detailed view into the effectiveness of CRISPR/Cas9 for genome editing in an economically important plant species.

CRISPR-Cas systems can be expressed in multiple ways, with different capabilities regarding tissue-specific expression, efficiency, and expression levels. Thus far, three expression strategies have been demonstrated in plants: mixed dual promoter systems, dual Pol II promoter systems, and single transcript unit (STU) systems. We explored a fourth strategy to express CRISPR-Cas9 in the model and crop plant, rice, where a bidirectional promoter (BiP) is used to express Cas9 and single guide RNA (sgRNA) in opposite directions. We first tested an engineered BiP system based on double-mini 35S promoter and an Arabidopsis enhancer, which resulted in 20.7% and 52.9% genome editing efficiencies at two target sites in T0 stable transgenic rice plants. We further improved the BiP system drastically by using a rice endogenous BiP, OsBiP1. The endogenous BiP expression system had higher expression strength and led to 75.9-93.3% genome editing efficiencies in rice T0 generation, when the sgRNAs were processed by either tRNA or Csy4. We provided a proof-of-concept study of applying BiP systems for expressing two-component CRISPR-Cas9 genome editing reagents in rice. Our work could promote future research and adoption of BiP systems for CRISPR-Cas-based genome engineering in plants.