Genome Editing In Wheat Research Articles

Improved biolistic transformation and genome editing in wheat by using trehalose for high osmotic treatment.

The tissue culture process is usually involved in gene transfer and genome editing in plants. Like other species, there is enormous variation among wheat genotypes in tissue culture response. In the rapidly advancing system of CRISPR/Cas9 for genome editing, particle bombardment has received increasing attention as a delivery method for a large amount of nucleic acids and RNA-protein complexes. However, the efficiency of transformation by particle bombardment has been low in wheat, and only a limited number of varieties have been transformed. In this study, replacement of maltose with trehalose as an osmolyte for high osmotic treatment for the protection of tissues from physical impacts improved callus formation in immature wheat embryos and efficiency of transformation and genome editing in varieties that are relatively poor in tissue culture response. The range of varieties amenable to biolistic transformation and genome editing may be expanded by this modification.

Enhancing wheat regeneration and genetic transformation through overexpression of TaLAX1

In the realm of genetically transformed crops, the process of plant regeneration holds utmost significance. However, the low regeneration efficiency of several wheat varieties currently restricts the use of genetic transformation for gene functional analysis and improved crop production. This research explores overexpression of TaLAX PANICLE1 (TaLAX1), which markedly enhances regeneration efficiency, thereby boosting genetic transformation and genome editing in wheat. Particularly noteworthy is the substantial increase in regeneration efficiency of common wheat varieties previously regarded as recalcitrant to genetic transformation. Our study shows that increased expression of TaGROWTH-REGULATING FACTOR (TaGRF) genes, alongside that of their co-factor, TaGRF-INTERACTING FACTOR 1 (TaGIF1), enhances cytokinin accumulation and auxin response, which may play pivotal roles in the improved regeneration and transformation of TaLAX1-overexpressing wheat plants. Overexpression of TaLAX1 homologs also significantly increases the regeneration efficiency of maize and soybean, suggesting that both monocot and dicot crops can benefit from this enhancement. Our findings shed light on a gene that enhances wheat genetic transformation and elucidate molecular mechanisms that potentially underlie wheat regeneration.

CRISPR/Cas9 genome editing in wheat: enhancing quality and productivity for global food security-a review.

Wheat (Triticum aestivum L.) is an important cereal crop that is grown all over the world for food and industrial purposes. Wheat is essential to the human diet due to its rich content of necessary amino acids, minerals, vitamins, and calories. Various wheat breeding techniques have been utilized to improve its quality, productivity, and resistance to biotic and abiotic stress impairing production. However, these techniques are expensive, demanding, and time-consuming. Additionally, these techniques need multiple generations to provide the desired results, and the improved traits could be lost over time. To overcome these challenges, researchers have developed various genome editing tools to improve the quality and quantity of cereal crops, including wheat. Genome editing technologies evolve quickly. Nowadays, single or multiple mutations can be enabled and targeted at specific loci in the plant genome, allowing controlled removal of undesirable features or insertion of advantageous ones. Clustered, regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) is a powerful genome editing tool that can be effectively used for precise genome editing of wheat and other crops. This review aims to provide a comprehensive understanding of this technology's potential applications to enhance wheat's quality and productivity. It will first explore the function of CRISPR/Cas9 in preserving the adaptive immunity of prokaryotic organisms, followed by a discussion of its current applications in wheat breeding.

Protoplast Isolation and Transfection in Wheat.

Wheat is one of the major staple crops around the world. A transient expression system is crucial for gene functional studies in wheat as stable transfection is still difficult in most cultivars. Protoplasts could serve as a versatile transient expression tool in wheat research. Here, we describe protocols for wheat protoplast isolation and transfection that are enabled by cellulase R-10 and macerozyme R-10 containing enzymatic solution and polyethylene glycol-mediated method, respectively. In addition, we show an example of efficiency evaluation of the emerging base editors in wheat protoplasts. These protocols are of wide use in both conventional gene functional analysis and reagent functionality evaluation of genome editing in wheat.

Current Status and Perspectives on the Application of CRISPR/Cas9 Gene-Editing System to Develop a Low-Gluten, Non-Transgenic Wheat Variety.

Wheat gluten contains epitopes that trigger celiac disease (CD). A life-long strict gluten-free diet is the only treatment accepted for CD. However, very low-gluten wheat may provide an alternative treatment to CD. Conventional plant breeding methods have not been sufficient to produce celiac-safe wheat. RNA interference technology, to some extent, has succeeded in the development of safer wheat varieties. However, these varieties have multiple challenges in terms of their implementation. Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease 9 (CRISPR/Cas9) is a versatile gene-editing tool that has the ability to edit immunogenic gluten genes. So far, only a few studies have applied CRISPR/Cas9 to modify the wheat genome. In this article, we reviewed the published literature that applied CRISPR/Cas9 in wheat genome editing to investigate the current status of the CRISPR/Cas9 system to produce a low-immunogenic wheat variety. We found that in recent years, the CRISPR/Cas9 system has been continuously improved to edit the complex hexaploid wheat genome. Although some reduced immunogenic wheat varieties have been reported, CRISPR/Cas9 has still not been fully explored in terms of editing the wheat genome. We conclude that further studies are required to apply the CRISPR/Cas9 gene-editing system efficiently for the development of a celiac-safe wheat variety and to establish it as a “tool to celiac safe wheat”.

Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture

Genome editing provides novel strategies for improving plant traits but mostly relies on conventional plant genetic transformation and regeneration procedures, which can be inefficient. In this study, we have engineered a Barley stripe mosaic virus–based sgRNA delivery vector (BSMV-sg) that is effective in performing heritable genome editing in Cas9-transgenic wheat plants. Mutated progenies were present in the next generation at frequencies ranging from 12.9% to 100% in three different wheat varieties, and 53.8%–100% of mutants were virus free. We also achieved multiplex mutagenesis in progeny using a pool of BSMV-sg vectors harboring different sgRNAs. Furthermore, we devised a virus-induced transgene-free editing procedure to generate Cas9-free wheat mutants by crossing BSMV-infected Cas9-transgenic wheat pollen with wild-type wheat. Our study provides a robust, convenient, and tissue culture–free approach for genome editing in wheat through virus infection.

Efficient genome editing in wheat using Cas9 and Cpf1 (AsCpf1 and LbCpf1) nucleases.

Genome editing can be used to create new wheat varieties with enhanced performance. Clustered regularly interspaced short palindromic repeat (CRISPR) is a powerful tool for knockout generation, precise modification, multiplex engineering, and the activation and repression of target genes. Targeted mutagenesis via RNA-guided genome editing using type II CRISPR-Cas9 is highly efficient in some plant species, but not in others. One possible solution is to use newly discovered variants of genome editing enzymes such as the class 2 system component Cpf1 (CRISPR from Prevotella and Francisella 1) in place of the more commonly used Cas9. We compared the editing efficiency of Cas9 and two Cpf1 orthologs, AsCpf1 (Acidaminococcus spp. BV3L6) and LbCpf1 (Lachnospiraceae bacterium ND2006) in wheat (Triticum aestivum). LbCpf1 had a higher editing efficiency for the target gene TaPDS than AsCpf1 and Cas9, and Cas9 induced more off-target mutations than AsCpf1 and LbCpf1, suggesting that CRISPR-LbCpf1 is a powerful genome editing tool for polyploid plants such as wheat.

A rapid method for detection of mutations induced by CRISPR/Cas9-based genome editing in common wheat.

Genome editing using CRISPR/Cas9 is useful for common wheat because common wheat has allohexaploid nature and it can induce mutations simultaneously in three homoeologous genes. Although Agrobacterium-mediated transformation has advantages in genome editing, it still has low efficiency and requires relatively long time in wheat. Therefore, the use of guide RNAs (gRNAs) with efficient mutagenesis in vivo is one of the critical factors for producing genome-edited mutant lines in a short time. In this study, we targeted three genes in common wheat and established a rapid method for detection of mutations induced by the biolistic transient expression system. Biolistic transient expression of the gRNAs and Cas9 was achieved in immature wheat embryos. Mutations were detected a week later using PCR-RFLP and verified by the sequencing of genomic clones. We confirmed several types of mutations that occurred at different rates depending on the target sequences. Furthermore, frequencies of mutations tended to be higher at the targets that were edited at higher rates in the plants transformed by Agrobacterium. These results show that this method of rapid detection of edited mutations could be used for variety of applications, such as screening of target sequences or modified vectors for efficient CRISPR/Cas9 genome editing in wheat.

CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat

Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops. High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat. The emerging effective genome editing tool, CRISPR/Cas9 system, makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles. To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat, we compared four different RNA polymerase (Pol) III promoters (TaU3p, TaU6p, OsU3p, and OsU6p) and three types of sgRNA scaffold in the protoplast system. We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective. The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles, whose orthologs, OsNP1 in rice and ZmIPE1 in maize, encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility. Triple homozygous mutations in TaNP1 genes result in complete male sterility. We further demonstrated that any one wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility. Taken together, this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.

WheatCRISPR: a web-based guide RNA design tool for CRISPR/Cas9-mediated genome editing in wheat

BackgroundCRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement.DescriptionWe have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184–191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR.ConclusionThe WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/.

GRNA validation for wheat genome editing with the CRISPR-Cas9 system

BackgroundThe CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate.ResultsThe seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat.ConclusionsWe have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.

Recent developments and applications of genetic transformation and genome editing technologies in wheat.

Wheat (Triticum aestivum) is a staple crop across the world and plays a remarkable role in food supplying security. Over the past few decades, basic and applied research on wheat has lagged behind other cereal crops due to the complex and polyploid genome and difficulties in genetic transformation. A breakthrough called as PureWheat was made in the genetic transformation of wheat in 2014 in Asia, leading to a noticeable progress of wheat genome editing. Due to this great achievement, it is predicated that wheat biotechnology revolution is arriving. Genome editing technologies using zinc finger nucleases, transcription activator-like effector nuclease, and clustered regularly interspaced short palindromic repeats-associated endonucleases (CRISR/Cas) are becoming powerful tools for crop modification which can help biologists and biotechnologists better understand the processes of mutagenesis and genomic alteration. Among the three genome editing systems, CRISR/Cas has high specificity and activity, and therefore it is widely used in genetic engineering. Generally, the genome editing technologies depend on an efficient genetic transformationsystem. In this paper, we summarize recent progresses and applications on genetic transformation and genome editing in wheat. We also examine the future aspects of genetic transformation and genome editing. We believe that the technologies for wheat efficient genetic engineering and functional studies will become routine with the emergence of high-quality genomic sequences.

CRISPR-based genome editing in wheat: a comprehensive review and future prospects.

CRISPR technology has vividly increased its applications in last five years for genome editing in a wide range of organisms from bacteria to plants. It is mostly applied in the field of mammalian research. This emerging versatile tool can be utilized in crop improvement by targeting various traits to increase economic value and adaptability of the crop species under changing climate. In plants, Arabidopsis and rice are the most studied plant species in genome editing through CRISPR technology. Wheat is lagging behind in the utilization of CRISPR based genome modifications. The hexaploid, large genome size and the recalcitrant nature in terms of tissue culture are the major obstacles for CRISPR utilization in wheat. Recently, the IWGSC released the high quality of reference genome for wheat which will greatly accelerate the application of CRISPR-based genome engineering in wheat and helps to resolve the global issue of food security in coming decades. The exogenous DNA-free improved mutants with CRISPR technology having desired traits will increase the productivity under biotic and abiotic stress conditions. To address complex traits involving multigene, recently developed multiplex genome editing toolkits can be used. This is a first review of its kind in which the practical utilization and updates on CRISPR validation in wheat along with its future prospects for use of this technology in wheat improvement are comprehensively discussed. Thus, the compiled information will immensely benefit the researchers for utilization of CRISPR system in wheat improvement across the globe.

Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing.

Summary CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co‐transferred plasmid backbone and low edit rate in wheat, a large T0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium‐delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain‐regulatory genes, TaCKX2‐1, TaGLW7, TaGW2, and TaGW8, in T0, T1, and T2 generation plants at an average edit rate of 10% without detecting off‐target mutations in the most Cas9‐active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160‐bp deletion in TaCKX2‐D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium‐delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.

Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat

The current application of genome editing to crop plants is limited to cultivars that are amenable to in vitro culture and regeneration. Here, we report an in planta genome-editing which does not require callus culture and regeneration. Shoot apical meristems (SAMs) contain a subepidermal cell layer, L2, from which germ cells later develop during floral organogenesis. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaGASR7 were bombarded into SAM-exposed embryos of imbibed seeds. Bombarded embryos showing transient GFP expression within SAM were selected and grown into adult plants. Mutations in the target gene were assessed in fifth-leaf tissue by cleaved amplified polymorphic sequence analysis. Eleven (5.2%) of the 210 bombarded plants carried mutant alleles, and the mutations of three (1.4%) of these were inherited in the next generation. Genotype analysis of T1 plants identified plants homozygous for the three homeologous genes, which were all derived from one T0 plant. These plants showed no detectable integration of the Cas9 and guide RNA genes, indicating that transient expression of CRISPR/Cas9 introduced the mutations. Together, our current method can be used to achieve in planta genome editing in wheat using CRISPR/Cas9 and suggests possible applications to other recalcitrant plant species and variations.

Development and Application of Genetic Engineering for Wheat Improvement

Wheat is one of the most important staple food crops of the world, and continuous genetic improvement is vital to meet the demands of the rapidly growing world population. Conventional breeding has led to the development of current high yielding wheat varieties, and recent achievements in genetic engineering are expected to augment conventional breeding to further increase production. Advances in genome sequencing and molecular breeding have increased the rate of gene discovery, leading to a need for highly efficient and robust transformation systems. Targeted genome editing will require efficient delivery of sequence-specific nucleases, such as zinc fingers (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases such as CRISPR-Cas9. Since the first report of fertile transgenic wheat in 1992, optimization of plant tissue culture techniques, DNA delivery methods, gene expression cassettes, and marker genes have led to reliable transformation protocols for a range of wheat model cultivars. However, like other cereal crops, wheat transformation has also been hampered by genotype effects. The limited range of transformable tissues in wheat is considered another challenge. Several excellent review papers have described the progress made towards developing robust genetic transformation systems for wheat, so we have focused our attention on a detailed analysis of selectable markers and promoters that have been used. The choice of selectable marker and promoter can dramatically influence the outcome of a transformation project. Both Agrobacterium tumefaciens and microprojectile-mediated transformation systems have been employed successfully for genetic transformation of wheat using genes of agronomic importance. Since improvement in agronomic traits of wheat will affect a sizeable population, we have provided an analysis of the progress made towards developing genetically superior wheat containing gene(s) of agronomic importance. Recent efforts on targeted genome editing in wheat are also discussed.

Genotyping genome-edited mutations in plants using CRISPR ribonucleoprotein complexes.

SummaryDespite the great achievements in genome editing, accurately detecting mutations induced by sequence‐specific nucleases is still a challenge in plants, especially in polyploidy plants. An efficient detection method is particularly vital when the mutation frequency is low or when a large population needs to be screened. Here, we applied purified CRISPR ribonucleoprotein complexes to cleave PCR products for genome‐edited mutation detection in hexaploid wheat and diploid rice. We show that this mutation detection method is more sensitive than Sanger sequencing and more applicable than PCR/RE method without the requirement for restriction enzyme site. We also demonstrate that this detection method is especially useful for genome editing in wheat, because target sites are often surrounded by single nucleotide polymorphisms. Using this screening method, we were also able to detect foreign DNA‐free tagw2 mutations induced by purified TALEN protein. Finally, we show that partial base editing mutations can also be detected using high‐fidelity SpCas9 variants or FnCpf1. The PCR/RNP method is low‐cost and widely applicable for rapid detection of genome‐edited mutation in plants.

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Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.