CRISPR/Cas systems: opportunities and challenges for crop breeding
11 May 2021-Plant Cell Reports (Springer Science and Business Media LLC)-Vol. 40, Iss: 6, pp 979-998
TL;DR: In this paper, the authors review the applications of CRISPR/Cas systems in crop breeding focusing on crop domestication, heterosis, haploid induction, and synthetic biology, and summarize the screening methods of C-Cas-induced mutations in crops.
Abstract: Increasing crop production to meet the demands of a growing population depends largely on crop improvement through new plant-breeding techniques (NPBT) such as genome editing. CRISPR/Cas systems are NPBTs that enable efficient target-specific gene editing in crops, which is supposed to accelerate crop breeding in a way that is different from genetically modified (GM) technology. Herein, we review the applications of CRISPR/Cas systems in crop breeding focusing on crop domestication, heterosis, haploid induction, and synthetic biology, and summarize the screening methods of CRISPR/Cas-induced mutations in crops. We highlight the importance of molecular characterization of CRISPR/Cas-edited crops, and pay special attentions to emerging highly specific genome-editing tools such as base editors and prime editors. We also discuss future improvements of CRISPR/Cas systems for crop improvement.
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TL;DR: In this paper, a review of green strategies and advanced technologies for sustainable and climate-smart agriculture is presented, and the gap between the results obtained in controlled experiments and those from application of these technologies in real field conditions (lab to field step) is also discussed.
Abstract: During the last years, a great effort has been dedicated at the development and employment of diverse approaches for achieving more stress-tolerant and climate-flexible crops and sustainable yield increases to meet the food and energy demands of the future. The ongoing climate change is in fact leading to more frequent extreme events with a negative impact on food production, such as increased temperatures, drought, soil salinization as well as invasive arthropod pests and diseases. In this review, diverse "green strategies" (e.g., chemical priming, root-associated microorganisms), and advanced technologies (e.g., genome editing, high-throughput phenotyping) are described on the basis of the most recent research evidence. Particularly, attention has been focused on the potential use in a context of sustainable and climate-smart agriculture (the so called "next agriculture generation") to improve plant tolerance and resilience to abiotic and biotic stresses. In addition, the gap between the results obtained in controlled experiments and those from application of these technologies in real field conditions (lab to field step) is also discussed. This article is protected by copyright. All rights reserved.
18 citations
29 Jul 2021
TL;DR: In this article, the detrimental effects of waterlogging on physiological and genetic mechanisms in four major cereal crops (rice, maize, wheat, and barley) were reviewed, including genes, genes, and quantitative trait loci associated with water-logging tolerance related traits.
Abstract: Waterlogging occurs when soil is saturated with water, leading to anaerobic conditions in the root zone of plants. Climate change is increasing the frequency of waterlogging events, resulting in considerable crop losses. Plants respond to waterlogging stress by adventitious root growth, aerenchyma formation, energy metabolism, and phytohormone signalling. Genotypes differ in biomass reduction, photosynthesis rate, adventitious roots development, and aerenchyma formation in response to waterlogging. We reviewed the detrimental effects of waterlogging on physiological and genetic mechanisms in four major cereal crops (rice, maize, wheat, and barley). The review covers current knowledge on waterlogging tolerance mechanism, genes, and quantitative trait loci (QTL) associated with waterlogging tolerance-related traits, the conventional and modern breeding methods used in developing waterlogging tolerant germplasm. Lastly, we describe candidate genes controlling waterlogging tolerance identified in model plants Arabidopsis and rice to identify homologous genes in the less waterlogging-tolerant maize, wheat, and barley.
16 citations
TL;DR: In this article , the authors discuss the exploitation of customized synthetic microbial communities in agricultural systems to develop more sustainable breeding strategies based on the implementation of multiple interactions between plants and their beneficial associated microorganisms.
15 citations
TL;DR: In this paper , the authors outline the current knowledge in alternative agricultural practices for achieving sustainability as well as policies and practices that need to be implemented for an equitable distribution of resources and food for achieving several goals in the UN 2030 Agenda for Sustainable Development.
Abstract: Despite world food production keeping pace with population growth because of the Green Revolution, the United Nations (UN) State of Food Security and Nutrition in the World 2022 Report indicates that the number of people affected by hunger has increased to 828 million with 29.3% of the global population food insecure, and 22% of children under five years of age stunted. Many more have low-quality, unhealthy diets and micronutrient deficiencies leading to obesity, diabetes, and other diet-related non-communicable diseases. Additionally, current agro-food systems significantly impact the environment and the climate, including soil and water resources. Frequent natural disasters resulting from climate change, pandemics, and conflicts weaken food systems and exacerbate food insecurity worldwide. In this review, we outline the current knowledge in alternative agricultural practices for achieving sustainability as well as policies and practices that need to be implemented for an equitable distribution of resources and food for achieving several goals in the UN 2030 Agenda for Sustainable Development. According to the UN Intergovernmental Panel on Climate Change, animal husbandry, particularly ruminant meat and dairy, accounts for a significant proportion of agricultural greenhouse gas (GHG) emissions and land use but contributes only 18% of food energy. In contrast, plant-based foods, particularly perennial crops, have the lowest environmental impacts. Therefore, expanding the cultivation of perennials, particularly herbaceous perennials, to replace annual crops, fostering climate-smart food choices, implementing policies and subsidies favoring efficient production systems with low environmental impact, empowering women, and adopting modern biotechnological and digital solutions can help to transform global agro-food systems toward sustainability. There is growing evidence that food security and adequate nutrition for the global population can be achieved using climate-smart, sustainable agricultural practices, while reducing negative environmental impacts of agriculture, including GHG emissions.
15 citations
TL;DR: An RNA‐Seq analysis identified critical genes in pollen development that were down‐regulated in flowers of eif4e/eIF4e plants, and suggested that eIF4E‐specific mRNA translation initiation is a limiting factor for male gametes formation in melon.
Abstract: Summary The cap‐binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap‐dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single‐nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non‐transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non‐mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild‐type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild‐type. An RNA‐Seq analysis identified critical genes in pollen development that were down‐regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E‐specific mRNA translation initiation is a limiting factor for male gametes formation in melon.
13 citations
References
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TL;DR: This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
12,865 citations
TL;DR: The power of the CRISPR-Cas9 technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders is illustrated.
Abstract: The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.
4,774 citations
TL;DR: E engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution.
Abstract: Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
3,384 citations
TL;DR: Recently devised sgRNA design rules are used to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results, and a metric to predict off-target sites is developed.
Abstract: CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.
2,866 citations
TL;DR: Adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA are described and a transfer RNA adenosine deaminase is evolved to operate on DNA when fused to a catalytically impaired CRISPR–Cas9 mutant.
Abstract: The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.
2,451 citations