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Multiples genome editing in foxtail millet using the CRISPR/Cas9 system. (A) Different strategies for simultaneously expressing multiple single guide RNA (sgRNAs). (B) Comparison of mutagenesis frequencies at four sgRNA target sites induced by single knockout constructs and multiple (MCTU-, tRNA-, and Csy4- based) knockout constructs in protoplast assay. (C) Sanger sequence chromatograms of quadruple mutations at the Dof4, BADH2, GBSS1, and IPK1 target sites of representative T0 plants. The positions in which indels occurred were indicated by black arrows.
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The genome editing toolbox based on CRISPR/Cas9 has brought revolutionary changes to agricultural and plant scientific research. With the development of stable genetic transformation protocols, a highly efficient genome editing system for foxtail millet (Setaria italica) is required. In the present study, we use the CRISPR/Cas9 single- and multi-ge...
Citations
... Induced site-directed mutations in the kafirin genes of sorghum by CRISPR/Cas9 improved protein digestibility and vitreous endosperm [139,140]. Herbicide-tolerant foxtail millet was developed by targeting two genes (acetolactate synthase (SiALS) and acetylcoenzyme A carboxylase (SiACC)) through CRISPR base editors (cytosine and adenosine base editors) [141]. The knockout of two carotenoid cleavage dioxygenase genes (SbCCD8a and SbCCD8b) reduced orobanchol production and parasite weed (in particular Striga) germination in sorghum [142]. ...
Citation: Maharajan, T.; Krishna, T.P.A.; Krishnakumar, N.M.; Vetriventhan, M.; Kudapa, H.; Ceasar, S.A. Role of Genome Sequences of Major and Minor Millets in Strengthening Food and Nutritional Security for Future Generations. Abstract: Millets are small-seeded cereals belonging to the family Poaceae. They are considered to be climate-resilient and future nutritional food cereals for humans. Millets are resistant to biotic and abiotic stressors compared to other major cereals and thrive in low-quality soils with little maintenance and less rainfall. The importance of millets is still not well known to many people due to the lack of popularity and cultivation in semi-arid tropics of Asia and Africa. The United Nations has declared 2023 as the International Year of Millets (IYM 2023) to promote millet cultivation and popularize their health benefits globally. A few years ago, the application of molecular biology was in its infancy in millets due to the unavailability of genome sequences. Genome sequences are available for most of the millets on NCBI and Phytozome databases. In this review, we discuss the details of genome sequences for millets, candidate genes identified from the native genome of millets. The current status of quantitative trait loci and genome-wide association studies in millets are also discussed. The utilization of millet genome sequences in functional genomics research and translating the information for crop improvement will help millet and non-millet cereals survive harsh environments in the future. Such efforts will help strengthen food security and reduce malnutrition worldwide in 2050.
... Induced site-directed mutations in the kafirin genes of sorghum by CRISPR/Cas9 improved protein digestibility and vitreous endosperm [139,140]. Herbicide-tolerant foxtail millet was developed by targeting two genes (acetolactate synthase (SiALS) and acetylcoenzyme A carboxylase (SiACC)) through CRISPR base editors (cytosine and adenosine base editors) [141]. The knockout of two carotenoid cleavage dioxygenase genes (SbCCD8a and SbCCD8b) reduced orobanchol production and parasite weed (in particular Striga) germination in sorghum [142]. ...
Citation: Maharajan, T.; Krishna, T.P.A.; Krishnakumar, N.M.; Vetriventhan, M.; Kudapa, H.; Ceasar, S.A. Role of Genome Sequences of Major and Minor Millets in Strengthening Food and Nutritional Security for Future Generations. Abstract: Millets are small-seeded cereals belonging to the family Poaceae. They are considered to be climate-resilient and future nutritional food cereals for humans. Millets are resistant to biotic and abiotic stressors compared to other major cereals and thrive in low-quality soils with little maintenance and less rainfall. The importance of millets is still not well known to many people due to the lack of popularity and cultivation in semi-arid tropics of Asia and Africa. The United Nations has declared 2023 as the International Year of Millets (IYM 2023) to promote millet cultivation and popularize their health benefits globally. A few years ago, the application of molecular biology was in its infancy in millets due to the unavailability of genome sequences. Genome sequences are available for most of the millets on NCBI and Phytozome databases. In this review, we discuss the details of genome sequences for millets, candidate genes identified from the native genome of millets. The current status of quantitative trait loci and genome-wide association studies in millets are also discussed. The utilization of millet genome sequences in functional genomics research and translating the information for crop improvement will help millet and non-millet cereals survive harsh environments in the future. Such efforts will help strengthen food security and reduce malnutrition worldwide in 2050.
... Cytosine and adenine base editors have demonstrated their efficacy in a variety of major crop species and model plants, allowing for precise modifications of genes linked to single nucleotide polymorphisms (SNPs) [33,86,87,171,187,191,218]. However, there is only one report on using base editors for crop improvement in foxtail millet, which employs base editing for precise base substitutions in the acetolactate synthase (ALS) and acetyl-coenzyme A carboxylase (ACC ) genes to enhance herbicide tolerance of the crop [93]. Despite this limited documentation, there is potential for extrapolating the knowledge gained from applying these tools to other plant species. ...
... In another multi-gene editing approach, three distinct multiplexing approaches were employed utilizing distinct promoters to drive the sgRNA expression viz. multicomponent transcriptional unit utilizing OsU3, TaU3, and AtU6 promoters, Csy4 method utilizing Panicum virgatum PvUbi promoter to drive expression of multiple sgRNAs separated by Csy4 hairpins and polycistronic tRNA-gRNA method utilizing OsU3 promoters to drive the expression of multiple sgRNAs [93]. ...
... In the case of millets, the trend of using ZmUbi constitutive promoter for expression of the nuclease gene is observed to be followed in the limited number of available gene editing studies in foxtail millet and green millet. In the case of foxtail millet, the multiplexing and the CBE/ ABE approach have employed the ZmUbi promoter to drive the nucleases [93] (Table 2). Similarly, the ZmUbi promoter is preferred for Cas9 expression in an attempt to edit foxtail millet [34,164,205,206] (Table 2) and green millet genes [10,189] (Table 2). ...
... T-DNA carrying cytosine base editor (CBE)/guide RNA (gRNA)-Cas12a/CRISPR RNA (crRNA)-green fluorescent protein (GFP) (Extended Data Fig. 1) is employed to co-edit the ALS gene, which encodes acetolactate synthase, and gene(s) of interest (GoI). Mutation in the ALS genes using CBE confers resistance to sulfonylurea herbicides such as chlorsulfuron in plants 8,13,[19][20][21][22][23][24][25][26] , thus providing a useful selection marker. The GoI are edited via Cas12a/ crRNA, whereas GFP enables screening of putative transgene-free (GFP-negative) transformants. ...
Transgene-free plant genome editing in the T0 generation is highly desirable but challenging1,2. Here we achieved such a goal using a co-editing strategy via Agrobacterium-mediated transient expression of cytosine base editor to edit ALS encoding acetolactate synthase to confer herbicide chlorsulfuron resistance as a selection marker, Cas12a/CRISPR RNA for editing gene(s) of interest, and green fluorescent protein for selecting transgene-free transformants. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 1.9% to 42.1% in tomato, tobacco, potato and citrus. This co-editing strategy is particularly useful for transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation.
... To increase the efficiency of this process, the DH production method should be precise and improved. However, while GE can potentially increase wheat production, this benefit is typically thwarted by low plant regeneration efficiency and a limited number of suitable transformable (Liang et al., 2022) genotypes in this species. Recently, a solution to this challenge has been suggested by Debernardi et al. (2020) through a combination of the CRISPR/Cas9 method with the TaGRF4-TaGIF1 approach, which increased the number of genotypes with sufficient transformation and regeneration efficiency. ...
... The DH induction frequency was observed to be up to 2.8% in the T 2 generation . Moreover, a multiplex gene knockout system has also been applied in foxtail millet by knocking out the FMBP, Dof4, BADH2, GBSS1, and IPK1 genes and developing homozygous plants through Agrobacterium-mediated transformation resulting in 100% mutation frequency in T 0 generations (Liang et al., 2022). ...
Cereal crops, including triticeae species (barley, wheat, rye), as well as edible cereals (wheat, corn, rice, oat, rye, sorghum), are significant suppliers for human consumption, livestock feed, and breweries. Over the past half-century, modern varieties of cereal crops with increased yields have contributed to global food security. However, presently cultivated elite crop varieties were developed mainly for optimal environmental conditions. Thus, it has become evident that taking into account the ongoing climate changes, currently a priority should be given to developing new stress-tolerant cereal cultivars. It is necessary to enhance the accuracy of methods and time required to generate new cereal cultivars with the desired features to adapt to climate change and keep up with the world population expansion. The CRISPR/Cas9 system has been developed as a powerful and versatile genome editing tool to achieve desirable traits, such as developing high-yielding, stress-tolerant, and disease-resistant transgene-free lines in major cereals. Despite recent advances, the CRISPR/Cas9 application in cereals faces several challenges, including a significant amount of time required to develop transgene-free lines, laboriousness, and a limited number of genotypes that may be used for the transformation and in vitro regeneration. Additionally, developing elite lines through genome editing has been restricted in many countries, especially Europe and New Zealand, due to a lack of flexibility in GMO regulations. This review provides a comprehensive update to researchers interested in improving cereals using gene-editing technologies, such as CRISPR/Cas9. We will review some critical and recent studies on crop improvements and their contributing factors to superior cereals through gene-editing technologies.
... Protoplasts have been used as transfection targets for GE tools in all nine crops covered by the current review except for cassava. The transfection of protoplasts is frequently used as a fast pre-screening system to test the efficacy of TALENs, Cas variants, or protospacers mutation frequency, with the aim of selecting the best target for obtaining mutated plants with standard transformation procedures [25,33,106,[109][110][111][112][113][114]. ...
Currently, the development of genome editing (GE) tools has provided a wide platform for targeted modification of plant genomes. However, the lack of versatile DNA delivery systems for a large variety of crop species has been the main bottleneck for improving crops with beneficial traits. Currently, the generation of plants with heritable mutations induced by GE tools mostly goes through tissue culture. Unfortunately, current tissue culture systems restrict successful results to only a limited number of plant species and genotypes. In order to release the full potential of the GE tools, procedures need to be species and genotype independent. This review provides an in-depth summary and insights into the various in vitro tissue culture systems used for GE in the economically important crops barley, wheat, rice, sorghum, soybean, maize, potatoes, cassava, and millet and uncovers new opportunities and challenges of already-established tissue culture platforms for GE in the crops.
... In foxtail millet, CRISPR/Cas9 mutagenesis has been reported in PDS gene (Lin et al., 2018). Recently, Liang et al. (2022) successfully created herbicide-tolerant (mutant) plants of foxtail millet through base editing (CBE and ABE) of two SiALS and SiACC genes and reported successful transmission of the mutations to next generation suggesting that it will now be possible to undertake research on multiples genome editing for improving millet crops. In the finger millet genome, no single-nucleotide polymorphism (SNP) has been discovered as of yet. ...
Nutritional insecurity has become a major concern for the ever growing world population. Millets are small-seeded cereal crops which are underutilized and neglected but possess abundant micronutrients and minerals. Millets are predominantly cultivated in Asia and Africa, and are an excellent alternative to major staple foods because of minimal water requirement, stress tolerance, adaptation to the marginal lands, and nutritional superiority compared to other cereal crops. Marker-assisted breeding with the next-generation sequencing approaches can speed up the genomic selection for the germplasm resource analysis, allele mining, QTL mapping, genome-wide marker-trait association, gene tagging, and fine-mapping for the millet improvement. With success, key cereal crops have been modified using genome editing techniques, but are limited to minor millets except for foxtail millet, though they have rich sources of nutrients. Lack of efficient transformation and genomic resources will have to gain momentum for successful genome editing in millets. There is a need to focus on advanced omics and genomics studies in millets to understand the molecular regulation of important traits and their transfer to other staple cereals for improvement. This chapter provide a thorough understanding of the use of genome editing, omics methods, and marker-assisted selection in minor millets to identify the necessary features for nutrient fortification and the creation of climate-resilient millets with improved targeted nutritional benefits.
... This enables the regulation of osmotic adjustment mechanisms, aiding in the plant's ability to cope with salt stress (Volkov et al., 2015) [41] . Several studies have demonstrated the positive effects of manipulating certain genes using these tools ( [46,19] . To explored the role of OsmiR535, a microRNA, in salt stress tolerance in rice. ...
... The CRISPR/Cas9 gene editing approach was used in sorghum by Li et al. (2018) to modify the k1C genes to reduce kafirin levels and improve protein quality and digestibility in the grain. In foxtail millet, CRISPR/Cas9 single-and multi-gene knockout systems were used to target the SiFMBP (Foxtail millet bran protein), SiDof4 (DNA-binding with one finger 1), SiBADH2 (Betaine aldehyde dehydrogenase 2), SiGBSS1 (Granule bound starch synthase 1), and SiIPK1 (Inositol-pentakisphosphate 2-kinase 1) genes to recover homozygous mutant plants through Agrobacterium-mediated genetic transformation (Liang et al., 2022) resulting in the mutagenesis frequency as high as 100% in T0 and subsequent generation with no off-target mutations at potential sites. Nutri-rich foxtail millet cultivars can be developed by employing the genome editing technology by targeting the genes for mineral contents. ...
The food that is consumed is the primary source of all the human nutrition needs. According to the World Health Organization, 49 known essential nutrients are necessary for sustaining human life. These can be broadly classified into six categories - water and energy, proteins, lipids-fat, macro-elements, micro-elements and vitamins. The primary source of all nutrients for people comes from agricultural products. The commercial agricultural systems world over mostly focused on increased productivity and profitability for farmers and agricultural industries, but never explicitly designed to promote human health. The concept that health should come from farm, not from pharmacy was fully forgotten during the agricultural revolution, which targeted only increased production and technological advancements leading to new agricultural practices. This resulted in selective breeding, increased dependence on the land and a more productive use of arable land, but decline in nutrition and a rise in infectious diseases. Malnutrition, both under (because of lack of availability or access) and over-nutrition (because of enhanced income levels) leading to obesity are the results of dysfunctional food systems that cannot supply all the nutrients and health-promoting factors. The human food pyramid suggests how the various food items need to be consumed in suitable proportions as per the body requirements of different nutrients.
... A decade ago, the genome sequences of two cultivars, Yugu1 and Zhanggu, were released (Bennetzen et al. 2012;Zhang et al. 2012), which laid the foundation for functional genomics studies. Recently, an efficient transformation method and new CRISPR/Cas9 genome-editing tools for foxtail millet have been established (Yang et al. 2020;Liang et al. 2022). Such developments promote foxtail millet as a novel model plant to understand the special developmental mechanism of C4 plants and the mechanism of plant stress tolerance. ...
