Gene Editing Improves the Agronomic Important Traits of Wheat – CRISPR-Cas9 and Cas12/Cpf1

Abstract

A hexaploid Wheat (Triticum aestivum L.) is the 3rd most important staple food crop with 15% caloric intake next to maize and rice in the world. The global attention for wheat improvement are still encouraging. However, the population growth and demand for food at this time and in the next years could not be balanced. Due to this, huge investments have been established and performed to improve the most important agronomic traits of wheat. Among the new molecular tools and techniques that have given a big emphasis as it will have many concerns is gene editing. Many gene editing tools have been reported and being implemented including Zinc finger nuclease, transcription activator-like effector nuclease, and clustered regularly interspaced short palindromic repeats associated Cas9/12 system for targeted gene editing. Among these, clustered regularly interspaced short palindromic repeats associated Cas9/12 systems are very accurate and widely used for targeted gene editing. By using CRISPR-Cas mediated gene editing technique, important traits of wheat include disease and pest resistance, better grain and flour quality, gluten-free trait, better nutritional value, nitrogen use efficiency, threshability, and other yield components and has been edited and improved. Therefore, the use of gene editing technologies for wheat as well as other important crops improvement was irreversible.

1. Introduction

The new approach and emerging technology of gene editing in crop plants and animals becomes a revolutionary science in the molecular era [1]. The conventional wheat crop genetic improvement or breeding progress has been described by the concept of genetic gain and measured by the difference between a selected population and its offspring population [2]. However, the global population growth is increasing at an increasing rate and is projected to reach 9.2billion in the coming 30 years [3, 4], and difficult to supply enough food and food products. Many studies suggested that improvement in genetic gain meet the growing population demand for agricultural products and food needs to utilize modern breeding techniques (tools and strategies) and platforms, implemented with improved agronomic practice, including improved field-based phenotyping with a better understanding of the genetic architecture of trait [2, 5].

Gene editing is among the new and growing technology of molecular science in crop improvement programs to improve the grain yield other agronomic important traits. The three most important gene-editing techniques widely used in the crop improvement program till this days are Zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats associated Cas9/12 (CRISPR-Cas9/12a) system for targeted gene editing [6, 7]. The development of CRISPR-based gene editing technologies recognizing distinct protospacer-adjacent motifs (PAMs), or having different spacer length/structure requirements broadens the range of possible genomic applications making them more preferred tools over ZFN and TALEN [8]. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein (CRISPR-Cas9/Cpf1) is a versatile, simple, and inexpensive system for precise sequence-specific modifications of DNA sequences including targeted mutagenesis for gene Knockout, single base substitution, and gene or allele replacement in vivo [6, 9]. CRISPR is a DNA fragment that contains non-contiguous short DNA repeats separated by spacers, which are snippets of varied sequences. CRISPR-associated (Cas) genes were anticipated to be related to CRISPR loci after they were found in the genome of Escherichia coli in 1987 [5].

Many genetic engineering activities have been done by different scientists across the world to get better traits related to grain yield, disease and pest resistance [3, 10], better grain and flour quality [11], gluten-free trait [12, 13], better nutritional value and nitrogen use efficiency [11] with the help of gene-editing tools. Similarly, in the USA Wang et al. [8] evaluated the natural and engineered variants of Cas12a (FnCas12a and LbCas12a) and Cas9 for their ability to induce mutations in endogenous genes controlling important agronomic traits in wheat. This review focuses on the application of the two CRISPR-Cas proteins (Cas9 and Cas12a) in the wheat crop improvement program. Therefore, this review highlights the current issues and advancements in wheat gene editing to improve the most important agronomic traits with aid of CRISPR-Cas proteins.

2. Gene editing for quality improvement

The higher demand for high-yielding varieties with premium grain quality in the continued economic development is a critical issue becomes increasing. Quality traits including grain yield, protein content, hectoliter weight, starch content are governed by many genes with a cumulative effect that is simultaneously affected by many factors, and it is more complicated in hexaploid wheat [11]. Improving this trait could not be a simple activity and not possible in conventional breeding or crop improvement program. Therefore, CRISPR Cas-mediated gene editing technologies have been used and created a great opportunity for allelic variations in a more faster and accurate manner [11]. Zhang et al. created and found allelic variations for grain hardiness, grain starch content, and dough color using genes of pinb, waxy, ppo, and psy in Fielder through Agrobacterium delivered CRISPR-Cas9 system. They had effectively obtained new wheat germplasms with better grain quality in hardiness, starch content, and dough color. Their improved grain quality wheat germplasms can be employed as donor parents in backcross breeding to improve the grain quality of premier wheat cultivars.

On the other hand, there is a need to have gluten-free wheat to overcome the risk of chronic disease (Coeliac disease (CD)). This disease is caused in genetically predisposed individuals by the ingestion of gluten proteins (gliadins and glutenins) from products of wheat, barley, and rye [12]. This human disease associated with wheat coeliac disease (CD) is an autoimmune reaction prevalent in 1–2% of the global population [12]. Even though gluten proteins are found in wheat, Jouanin et al. reported RNA interference (RNAi) silencing to down-regulate gliadin families which are capable of causing Coeliac disease. Therefore, an essential and strict lifelong treatment of CD is the consumption of gluten-free food sources and avoidance of gluten-containing products from wheat, rye, barley, and, in a rare case, oats [13].

3. Gene editing for disease resistance

Crop production is multi-task activity and is easily affected by many contributing factors for yield and quality reduction of the produce. Among the factors that greatly affect the quality and grain yield of wheat are biotic agents (pest, disease) and abiotic agents especially fertilization, soil acidity, drought, and cold stress besides its genetic potential. Although the conventional approach of crop protection (chemical and cultural) activities has been applied to protect the crop, the numbers, as well as the type of reported diseases and pests, become increasing [3]. Moreover, these classical breeding approaches to develop pest and disease-resistant varieties are laborious, cost-intensive, and not efficient. Therefore, the new technology and approach recently introduced gene editing via the CRISPR-Cas system has been utilized to protect the crops from pests and pathogens and to enhance disease and pest resistance among different crop plants i.e. Wheat, Rice, Cocoa, Tomato, and Grape [3]. In a similar study, fungal disease powdery mildew (Blumeria graminis f.sp. tritici (Bgt)) resistant gene (TaEDR1) is successfully introduced from Arabidopsis thaliana using gene editing via CRISPR-Cas9 technology to wheat [14]. In their study, they have cloned Triticum aestivum enhanced disease resistance-1 gene (TaEDR1) in hexaploid wheat and showed the knockdown TaEDR1 mutants from VIGs or RNAi increased resistance to virulent Bgt isolates. Then they have generated wheat edr1 plants by simultaneous modification of three homologs of TaEDR1 with the help of CRISPR Cas9 technology. Then, they have got wheat genotypes carrying TaEDR1 plants which did not exhibit mildew-induced cell death/disease symptoms. This candidate gene could be very important in the crop improvement program of wheat to overcome and reduce the problem of powdery mildew. Likewise, Brauer et al. [15] in Canada reported that gene editing of deoxynivalenol-induced transcription factor confers resistance to Fusarium head blight disease (Fusarium graminearum) in wheat. Therefore, gene editing has played a significant role in critically editing, improving, and developing candidate disease-resistant genes for wheat.

4. Gene editing for better nitrogen use efficiency

In the era of the green revolution, we remember what Norman Borlaug has contributed by developing a semi-dwarf wheat genotype that has a great efficiency for fertilizer response and then provides a higher grain yield. Although the semi-dwarf wheat type responded better than the older one, the crop has a great genetic potential to give more yield if its nitrogen use efficiency is edited using the molecular breeding technique CRISPR-Cas system. Moreover, scientists reported that the wheat crop has 40% nitrogen use efficiency whereas the remaining amount is released to the environment via leaching or volatilization [11]. To improve the NUE of wheat Zhang et al. [16] isolated and characterized three TaARE1 homoeologs from the elite Chinese winter wheat cultivar ZM and tare1 transgene-free mutant lines with partial or triple-null alleles. Under hydroponic conditions, all transgene-free mutant lines demonstrated greater tolerance to N-deficit or starvation (Figures 1 and 2), as well as delayed senescence and higher grain yield in a field experiment under normal growth circumstances (Figure 2). When compared to the wild-type control, the AABBdd and aabbDD mutant lines had considerably improved nitrogen use efficiency, postponed senescence, and increased grain production without showing any growth abnormalities (Figure 2). For the first time, they were able to create novel wheat germplasm with better NUE and yield potential by modifying TaARE1 by genome editing tool.

Figure 1.

Illustrates root morphology, root/shoot ratio, and chlorophyll content of taare1 mutant lines compared to the wild-type control. (a) Root morphology of wild-type and different taare1 mutant lines under N deficiency (0 mM NH4NO3) hydroponic condition (scale bars = 5 cm). (b) Root morphology of wild-type and different taare1 mutant lines under N supply (1.5 mM NH4NO3) hydroponic condition (scale bars = 5 cm). (c) Root/shoot ratio of wild-type and different taare1 mutant lines under different concentrations of N (0 mM NH4NO3,0.5 mM NH4NO3,1.0 mM NH4NO3, and 1.5 mM NH4NO3) hydroponic con- (d) Quantification of chlorophyll content in wild-type and different taare1 mutant lines under different concentrations of N (0 mM NH4NO3,0.5 mM NH4NO3,1.0 mM NH4NO3, and 1.5 mM NH4NO3) hydroponic conditions ([11], Journal of Integrative Plant Biology).

Figure 2.

Illustrates phenotypes of wild-type and different taare1 mutant lines in the field. (a) Plant phenotypes of wild-type and different taare1 mutant lines at the dough stage (scale bars = 10 cm). (b) Phenotypes of flag leaves of wild type and different taare1 mutant lines at the dough stage (scale bars = 5 cm). (c) Plant phenotypes of wild-type and different taare1 mutant lines at the dough stage in the field. (d) Plant phenotypes of wild-type and different taare1 mutant lines at the kernel ripe stage (scale bars = 10 cm). (e) Spike phenotypes of wild-type and different taare1 mutant lines at the kernel ripe stage (scale bars = 5 cm). (f) Grain size and appearance in wild-type and different taare1 mutant lines at the kernel ripe stage. The grains were aligned to illustrate grain length (a) and grain width (b) between wild-type and mutant lines (scale bars = 1 cm) ([11], Journal of Integrative Plant Biology).

5. Gene editing for yield component traits

Understanding the genetic basis of yield component traits in major crop plants holds a great promise to improve and utilize yield potential by allowing breeders to make informed decisions. Then, by assembling beneficial allelic combinations, it is possible to create new improved varieties [17]. Gene editing of the wheat homologs of TONNEAU1-recruiting motif encoding gene affects grain shape and weight in wheat by using CRISPR-Cas9 technology [18]. Likewise, Wang et al. showed that the CRISPR-Cas9 gene editing of TaGW7, a homolog of OsGW7 encoding a TONNEAU1-recruiting motif (TRM) protein affects grain shape and weight in allohexaploid wheat. Moreover, Wang et al. [8] also effectively used CRISPR LbCase12a-MGE gene-editing tool to generate heritable mutations in a wheat gene that controls grain size and weight. They found that utilizing altered Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that can identify TATV and NG PAMs respectively, the range of editable loci in a wheat genome may be further broadened with Cas9 NG indicating greater editing efficiency on targets with a typical PAMs than xCas9 (Figure 3).

Figure 3.

The DNA sequence and phenotypic differences between edited and unedited lines. Source: Zhang et al. [11].

6. Gene editing for haploid wheat development

Wheat has two species cultivated in the world i.e. bread wheat (Triticum aestivum L.) and durum wheat (Triticum durum or Triticum turgidum subspe. Durum.) [19]. The bread wheat species is allohexaploid, (2n = 6x = 42) consists of three sub-genomes (A, B and G), and is cultivated for the purpose of bread because of lower gluten content. Whereas durum wheat is tetraploid, (2n = 4x = 28) has two sub-genomes (A and B) lacking D and is cultivated for pasta and macaroni. In the era of molecular breeding and omics crop improvement, scientists come up with the development of haploid paternal wheat. Conventional breeding of the most important cereal crops (maize, wheat, and rice) is based on the genetic mating of different parents with distinct traits to develop a single crop variety having desirable allelic combination may take also 8–10 years [20]. However, gene editing (GE) with haploid induction (HI) was successfully performed on (T. aestivum L., Zea mays L., Hordeum vulgare L., Brassica napus L) and possible to develop pure homozygous DH genotypes within two generations (Figure 4) [17].

Figure 4.

Simple steps of Haploid inducer mediated editing (HIME) on maize crop.

Amin & Safwat [21], developed 120 Doubled haploid spring wheat genotypes from a cross of F1 of cross-pollination between wheat (Triticum aestivum L.) and millet (Pennisetum glaucum) with Indian cultivar (Kharchia) and Egyptian cultivar (Sakha 93). In this study, under normal conditions, agronomic traits of DH genotype traits (flowering time, number of spikelets per spike, plant height, spike length, and thousand seeds weight) revealed better performances. Therefore, to get the benefit of better performance of agronomic traits of wheat, doubled haploid (DH) genotype development is becoming a quick way (not more than 2 generations) of crop improvement [20].

In the conventional double haploid development, haploids may be induced either in vitro or in vivo methods [20]. Therefore, while performing the in vitro method of haploid production, isolated microspore culture, as well as an anther and ovary/ovule culture, needs colchicine/charcoal treatment and culturing the cell in the Petri-dish for the chromosome doubling but current in vivo methods are based on the modification of histone molecule H3 (CENH3). The conventional haploid development method takes a long time (6 years) than the haploid inducer mediated editing technology (Figure 5).

Figure 5.

The conventional haploid development procedure. Source: Bhowmik & Bilichak [20].

7. Other agronomic trait improvements of wheat

Free threshing in wheat is an advantage and leads to the selection of the domesticated Q allele, which is now present in almost all modern wheat varieties [22]. A study conducted by Liu et al. [23] to see the regulation mechanism of and improve wheat spike and threshability using CRISPR-Cas9 obtained homozygous plants in the F1 generation with loss of function of only TaAQ or TaDq and simultaneous loss of function of TaAQ and TaDq to analyze the effect of these genes on wheat spikes and floret shapes. Then, two genes of TaAQ and TaDq were edited using CRISPR-Cas9 and resulted improved spike morphogenesis and grain threshability. This shows that the TaQ gene families are very important in the improvement of different traits of wheat. In wheat, the loss of function mutant of the (AP2) like transcription factor the Q gene changed the flowering time and spike architecture. Because of the benefit of free threshing in wheat, the domesticated wheat allele Q was chosen, and it is now found in nearly all current wheat cultivars. The domesticated allele Q confers a free threshing trait and a subcompact (i.e. partially compact) inflorescence (spike), while the pre-domesticated allele q, encodes an AP2 transcription factor [22].

8. Conclusion

The global demand for food is increasing at an alarming rate because of the population growth as expected to reach greater than 9 billion in 2050. It is difficult to supply enough food and feed this much population now and coming 30 years using the usual and conventional crop improvement technique and approach. Scientists and different organizations in the world performed different research activities and developed new and novel tools, procedures as well as protocols in wheat crop improvement. In this review manuscript, the gene-editing tools (CRISPR Cas systems) role and advancement were covered and highlighted. Based on this, many findings reported that CRISPR-Cas9 and 12 systems have been successfully used and implemented to improve agronomic important traits of wheat crop. Mainly, traits related to grain yield, disease and pest resistance, better grain, and flour quality, gluten-free trait, better nutritional value, and nitrogen use efficiency were improved with the help of gene editing tools especially CRISPR-Cas9 and 12 (Cpf1). Surprisingly, gene editing has been successfully implemented in the doubled haploid production and reduced the time required for fixing the trait by 6 years than the conventional method.

9. Future prospects

Although gene-editing techniques are used to improve the qualitative and quantitative traits of wheat, still the wheat genetic yield potential improvement is difficult because of its polygenic and polyploidy nature. Grain yield and most quality traits sometimes may not be improved simultaneously due to their indirect association. Moreover, the science of biotechnology is still growing and needs time, skill, and technology to explore the association of each trait with the grain yield of wheat since the ultimate goal of any crop improvement is an economic yield of the crop. Therefore, for the future scientists and organizations in the world shall create the technology used to detect multiple specific regions of DNA sequences at a time and improve by pyramiding the genes. Finally, the gene-editing technologies had the best features of no risk of chemicals like colchicine and short (2 years) doubled haploid wheat genotype development.

Authors’ contributions

The first author drafted the manuscript, summarization of ideas, interpretation of the data, critical reviewing, synthesizing, and revision. The 2nd author’s contribution is the critical evaluation of the manuscript and providing critical comments.

Funding

Not applicable.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics approval and consent to participate

Not applicable. Because I have not used the human research data for this paper.

Availability of data and material

This review is done by collecting original published research articles and I have cited the authors for each idea and data taken from their article and included in the reference list.

Abbreviations

UNDESA	United Nations Department of Economic and Social Affairs
TALEN	Transcription Factor Like Effector Nuclease
CRISPR	Clustered Regularly Interspaced Short Palindromic Repeats
ZFN	Zink Finger Nuclease
CD	Coeliac Disease
TaEDR1	Triticum aestivum enhanced Disease Resistance gene1
NUE	Nitrogen Use Efficiency
VIGs	Virus Induced Gene Silencing
Bgt	Blumera Graminis titici