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    SgRNA engineering for improved genome editing and expanded functional assays. Current opinion in biotechnology The CRISPR/Cas system has been established as the most powerful and practical genome engineering tool for both fundamental researches and biotechnological applications. Great efforts have been devoted to engineering the CRISPR system with better performance and novel functions. As an essential component, single guide RNAs (sgRNAs) have been extensively designed and engineered with desirable functions. This review highlights representative studies that optimize the sgRNA nucleotide sequences for improved genome editing performance (e.g. activity and specificity) as well as add extra aptamers and end extensions for expanded CRISPR-based functional assays (e.g. transcriptional regulation, genome imaging, and prime editor). The perspectives for further sgRNA engineering to establish more powerful and versatile CRISPR/Cas systems are also discussed. 10.1016/j.copbio.2022.102697
    History of genome editing: From meganucleases to CRISPR. Tröder Simon E,Zevnik Branko Laboratory animals CRISPR-mediated genome editing has undoubtedly revolutionized genetic engineering of animals. With the ability for virtually unlimited modification of almost any genome it is easy to forget which amazing discoveries paved the way for this ground-breaking technology. Here, we summarize the history of genome editing platforms, starting from enhanced integration of foreign DNA by meganuclease-mediated double-strand breaks to CRISPR/Cas9, the leading technology to date, and its re-engineered variants. 10.1177/0023677221994613
    A Critical Review: Recent Advancements in the Use of CRISPR/Cas9 Technology to Enhance Crops and Alleviate Global Food Crises. Rasheed Adnan,Gill Rafaqat Ali,Hassan Muhammad Umair,Mahmood Athar,Qari Sameer,Zaman Qamar U,Ilyas Muhammad,Aamer Muhammad,Batool Maria,Li Huijie,Wu Ziming Current issues in molecular biology Genome editing (GE) has revolutionized the biological sciences by creating a novel approach for manipulating the genomes of living organisms. Many tools have been developed in recent years to enable the editing of complex genomes. Therefore, a reliable and rapid approach for increasing yield and tolerance to various environmental stresses is necessary to sustain agricultural crop production for global food security. This critical review elaborates the GE tools used for crop improvement. These tools include mega-nucleases (MNs), such as zinc-finger nucleases (ZFNs), and transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Specifically, this review addresses the latest advancements in the role of CRISPR/Cas9 for genome manipulation for major crop improvement, including yield and quality development of biotic stress- and abiotic stress-tolerant crops. Implementation of this technique will lead to the production of non-transgene crops with preferred characteristics that can result in enhanced yield capacity under various environmental stresses. The CRISPR/Cas9 technique can be combined with current and potential breeding methods (e.g., speed breeding and omics-assisted breeding) to enhance agricultural productivity to ensure food security. We have also discussed the challenges and limitations of CRISPR/Cas9. This information will be useful to plant breeders and researchers in the thorough investigation of the use of CRISPR/Cas9 to boost crops by targeting the gene of interest. 10.3390/cimb43030135
    Genome-wide identification studies - A primer to explore new genes in plant species. Safder I,Shao G,Sheng Z,Hu P,Tang S Plant biology (Stuttgart, Germany) Genome data have accumulated rapidly in recent years, doubling roughly after every 6 months due to the influx of next-generation sequencing technologies. A plethora of plant genomes are available in comprehensive public databases. This easy access to data provides an opportunity to explore genome datasets and recruit new genes in various plant species not possible a decade ago. In the past few years, many gene families have been published using these public datasets. These genome-wide studies identify and characterize gene members, gene structures, evolutionary relationships, expression patterns, protein interactions and gene ontologies, and predict putative gene functions using various computational tools. Such studies provide meaningful information and an initial framework for further functional elucidation. This review provides a concise layout of approaches used in these gene family studies and demonstrates an outline for employing various plant genome datasets in future studies. 10.1111/plb.13340
    Gene editing advances on all fronts. O'Leary Karen Nature medicine 10.1038/s41591-021-01607-z
    Current advances in overcoming obstacles of CRISPR/Cas9 off-target genome editing. Aquino-Jarquin Guillermo Molecular genetics and metabolism CRISPR/Cas9-based technology has revolutionized biomedical research by providing a high-fidelity gene-editing method, foreshadowing a significant impact on the therapeutics of many human genetic disorders previously considered untreatable. However, off-target events represent a critical hurdle before genome editing can be fully established in clinical practice. This mini-review recapitulates some recent advances for detecting and overcoming off-target effects mediated by the CRISPR/Cas9 system that could increase the likelihood of clinical success of the CRISPR-based approaches. 10.1016/j.ymgme.2021.08.002
    CRISPR: History and perspectives to the future. Kozovska Z,Rajcaniova S,Munteanu P,Dzacovska S,Demkova L Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie This review summarizes the information about the history and future of the CRISPR/Cas9 method. Genome editing can be perceived as a group of technologies that allow scientists to change the DNA of an organism. These technologies involve the deletion, insertion, or modification of the genome at a specific site in a DNA sequence. Gene therapy in humans has a perspective to be used to eliminate the gene responsible for a particular genetic disorder. The review focuses on the key elements of this promising method and the possibility of its application in the treatment of cancer and genetic diseases. 10.1016/j.biopha.2021.111917
    Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering. Huang Teng-Kuei,Puchta Holger Transgenic research In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage. 10.1007/s11248-021-00238-x
    Preface: Genome editing in plants. Christou Paul,Dhingra Amit,Slamet-Loedin Inez H,Oliveira Margarida,Chakraborty Supriya,Buyel Johannes,Stoger Eva,Schillberg Stefan,Orzaez Diego,Quemada Hector Transgenic research 10.1007/s11248-021-00268-5
    Recent advances of genome editing and related technologies in China. Sun Wen,Wang Haoyi Gene therapy Genome editing is a powerful tool, enabling scientists to alter DNA sequence at virtually any genome locus in any species. Different technologies have been developed employing programmable nucleases including meganuclease, zinc-finger nucleases, transcription activator-like effector nucleases, and most recently CRISPR-Cas systems. Chinese research groups are making important contributions at an increasing speed in genome editing field in recent years. In this review, we summarize recent progress made by Chinese scientists on the technological development of genome editing and beyond, focusing on the optimization and expanded application of existing genome editing tools, as well as the exploration of novel proteins as potential genome editing tools. 10.1038/s41434-020-0181-5
    Base Editing in Plants: Applications, Challenges, and Future Prospects. Azameti Mawuli K,Dauda Wadzani Palnam Frontiers in plant science The ability to create targeted modifications in the genomes of plants using genome editing technologies has revolutionized research in crop improvement in the current dispensation of molecular biology. This technology has attracted global attention and has been employed in functional analysis studies in crop plants. Since many important agronomic traits are confirmed to be determined by single-nucleotide polymorphisms, improved crop varieties could be developed by the programmed and precise conversion of targeted single bases in the genomes of plants. One novel genome editing approach which serves for this purpose is base editing. Base editing directly makes targeted and irreversible base conversion without creating double-strand breaks (DSBs). This technology has recently gained quick acceptance and adaptation because of its precision, simplicity, and multiplex capabilities. This review focuses on generating different base-editing technologies and how efficient they are in editing nucleic acids. Emphasis is placed on the exploration and applications of these base-editing technologies to enhance crop production. The review also highlights the drawbacks and the prospects of this new technology. 10.3389/fpls.2021.664997
    Knowing when to talk? Plant genome editing as a site for pre-engagement institutional reflexivity. Public understanding of science (Bristol, England) Citizen and stakeholder engagement is frequently portrayed as vital for socially accountable science policy but there is a growing understanding of how institutional dynamics shape engagement exercises in ways that prevent them from realising their full potential. Limited attention has been devoted to developing the means to expose institutional features, allow policy-makers to reflect on how they will shape engagement and respond appropriately. Here, therefore, we develop and test a methodological framework to facilitate pre-engagement institutional reflexivity with one of the United Kingdom's eminent science organisations as it grappled with a new, high-profile and politicised technology, genome editing. We show how this approach allowed policy-makers to reflect on their institutional position and enrich decision-making at a time when they faced pressure to legitimate decisions with engagement. Further descriptions of such pre-engagement institutional reflexivity are needed to better bridge theory and practice in the social studies of science. 10.1177/0963662521999796
    Targeted genome editing of plants and plant cells for biomanufacturing. Buyel J F,Stöger E,Bortesi L Transgenic research Plants have provided humans with useful products since antiquity, but in the last 30 years they have also been developed as production platforms for small molecules and recombinant proteins. This initially niche area has blossomed with the growth of the global bioeconomy, and now includes chemical building blocks, polymers and renewable energy. All these applications can be described as "plant molecular farming" (PMF). Despite its potential to increase the sustainability of biologics manufacturing, PMF has yet to be embraced broadly by industry. This reflects a combination of regulatory uncertainty, limited information on process cost structures, and the absence of trained staff and suitable manufacturing capacity. However, the limited adaptation of plants and plant cells to the requirements of industry-scale manufacturing is an equally important hurdle. For example, the targeted genetic manipulation of yeast has been common practice since the 1980s, whereas reliable site-directed mutagenesis in most plants has only become available with the advent of CRISPR/Cas9 and similar genome editing technologies since around 2010. Here we summarize the applications of new genetic engineering technologies to improve plants as biomanufacturing platforms. We start by identifying current bottlenecks in manufacturing, then illustrate the progress that has already been made and discuss the potential for improvement at the molecular, cellular and organism levels. We discuss the effects of metabolic optimization, adaptation of the endomembrane system, modified glycosylation profiles, programmable growth and senescence, protease inactivation, and the expression of enzymes that promote biodegradation. We outline strategies to achieve these modifications by targeted gene modification, considering case-by-case examples of individual improvements and the combined modifications needed to generate a new general-purpose "chassis" for PMF. 10.1007/s11248-021-00236-z
    The CRISPR/Cas9 revolution continues: From base editing to prime editing in plant science. Li Yan,Li Wenjing,Li Jun Journal of genetics and genomics = Yi chuan xue bao The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a revolutionary technology that could generate targeted mutants, has facilitated notable advances in plant science. Genome editing with CRISPR/Cas9 has gained great popularity and enabled several technical breakthroughs. Herein, we briefly introduce the CRISPR/Cas9, with a focus on the latest breakthroughs in precise genome editing (e.g., base editing and prime editing), and we summarize various platforms that developed to increase the editing efficiency, expand the targeting scope, and improve the specificity of base editing in plants. In addition, we emphasize the recent applications of these technologies to plants. Finally, we predict that CRISPR/Cas9 and CRISPR/Cas9-based genome editing will continue to revolutionize plant science and provide technical support for sustainable agricultural development. 10.1016/j.jgg.2021.05.001
    Genome editing for plant research and crop improvement. Zhan Xiangqiang,Lu Yuming,Zhu Jian-Kang,Botella Jose Ramon Journal of integrative plant biology The advent of clustered regularly interspaced short palindromic repeat (CRISPR) has had a profound impact on plant biology, and crop improvement. In this review, we summarize the state-of-the-art development of CRISPR technologies and their applications in plants, from the initial introduction of random small indel (insertion or deletion) mutations at target genomic loci to precision editing such as base editing, prime editing and gene targeting. We describe advances in the use of class 2, types II, V, and VI systems for gene disruption as well as for precise sequence alterations, gene transcription, and epigenome control. 10.1111/jipb.13063
    Genome editing for resistance against plant pests and pathogens. Rato Cláudia,Carvalho Miguel F,Azevedo Cristina,Oblessuc Paula Rodrigues Transgenic research The conventional breeding of crops struggles to keep up with increasing food needs and ever-adapting pests and pathogens. Global climate changes have imposed another layer of complexity to biological systems, increasing the challenge to obtain improved crop cultivars. These dictate the development and application of novel technologies, like genome editing (GE), that assist targeted and fast breeding programs in crops, with enhanced resistance to pests and pathogens. GE does not require crossings, hence avoiding the introduction of undesirable traits through linkage in elite varieties, speeding up the whole breeding process. Additionally, GE technologies can improve plant protection by directly targeting plant susceptibility (S) genes or virulence factors of pests and pathogens, either through the direct edition of the pest genome or by adding the GE machinery to the plant genome or to microorganisms functioning as biocontrol agents (BCAs). Over the years, GE technology has been continuously evolving and more so with the development of CRISPR/Cas. Here we review the latest advancements of GE to improve plant protection, focusing on CRISPR/Cas-based genome edition of crops and pests and pathogens. We discuss how other technologies, such as host-induced gene silencing (HIGS) and the use of BCAs could benefit from CRISPR/Cas to accelerate the development of green strategies to promote a sustainable agriculture in the future. 10.1007/s11248-021-00262-x
    Genome editing in fruit, ornamental, and industrial crops. Ramirez-Torres Fabiola,Ghogare Rishikesh,Stowe Evan,Cerdá-Bennasser Pedro,Lobato-Gómez Maria,Williamson-Benavides Bruce A,Giron-Calva Patricia Sarai,Hewitt Seanna,Christou Paul,Dhingra Amit Transgenic research The advent of genome editing has opened new avenues for targeted trait enhancement in fruit, ornamental, industrial, and all specialty crops. In particular, CRISPR-based editing systems, derived from bacterial immune systems, have quickly become routinely used tools for research groups across the world seeking to edit plant genomes with a greater level of precision, higher efficiency, reduced off-target effects, and overall ease-of-use compared to ZFNs and TALENs. CRISPR systems have been applied successfully to a number of horticultural and industrial crops to enhance fruit ripening, increase stress tolerance, modify plant architecture, control the timing of flower development, and enhance the accumulation of desired metabolites, among other commercially-important traits. As editing technologies continue to advance, so too does the ability to generate improved crop varieties with non-transgenic modifications; in some crops, direct transgene-free edits have already been achieved, while in others, T-DNAs have successfully been segregated out through crossing. In addition to the potential to produce non-transgenic edited crops, and thereby circumvent regulatory impediments to the release of new, improved crop varieties, targeted gene editing can speed up trait improvement in crops with long juvenile phases, reducing inputs resulting in faster market introduction to the market. While many challenges remain regarding optimization of genome editing in ornamental, fruit, and industrial crops, the ongoing discovery of novel nucleases with niche specialties for engineering applications may form the basis for additional and potentially crop-specific editing strategies. 10.1007/s11248-021-00240-3
    Construct design for CRISPR/Cas-based genome editing in plants. Hassan Md Mahmudul,Zhang Yingxiao,Yuan Guoliang,De Kuntal,Chen Jin-Gui,Muchero Wellington,Tuskan Gerald A,Qi Yiping,Yang Xiaohan Trends in plant science CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing. 10.1016/j.tplants.2021.06.015
    Genome editing of polyploid crops: prospects, achievements and bottlenecks. Schaart Jan G,van de Wiel Clemens C M,Smulders Marinus J M Transgenic research Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted. 10.1007/s11248-021-00251-0
    Recent advances in CRISPR technologies for genome editing. Song Myeonghoon,Koo Taeyoung Archives of pharmacal research The discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, and its development into a set of powerful tools for manipulating the genome, has revolutionized genome editing. Precise, targeted CRISPR/Cas-based genome editing has become the most widely used platform in organisms ranging from plants to animals. The CRISPR/Cas system has been extensively modified to increase its efficiency and fidelity. In addition, the fusion of various protein motifs to Cas effector proteins has facilitated diverse set of genetic manipulations, such as base editing, transposition, recombination, and epigenetic regulation. The CRISPR/Cas system is undergoing continuous development to overcome current limitations, including off-target effects, narrow targeting scope, and issues associated with the delivery of CRISPR components for genome engineering and therapeutic approaches. Here, we review recent progress in a diverse array of CRISPR/Cas-based tools. We also describe limitations and concerns related to the use of CRISPR/Cas technologies. 10.1007/s12272-021-01336-4
    Genome editing reagent delivery in plants. Ghogare Rishikesh,Ludwig Yvonne,Bueno Gela Myan,Slamet-Loedin Inez H,Dhingra Amit Transgenic research Genome editing holds the potential for rapid crop improvement to meet the challenge of feeding the planet in a changing climate. The delivery of gene editing reagents into the plant cells has been dominated by plasmid vectors delivered using agrobacterium or particle bombardment. This approach involves the production of genetically engineered plants, which need to undergo regulatory approvals. There are various reagent delivery approaches available that have enabled the delivery of DNA-free editing reagents. They invariably involve the use of ribonucleoproteins (RNPs), especially in the case of CRISPR/Cas9-mediated gene editing. The explant of choice for most of the non-DNA approaches utilizes protoplasts as the recipient explant. While the editing efficiency is high in protoplasts, the ability to regenerate individual plants from edited protoplasts remains a challenge. There are various innovative delivery approaches being utilized to perform in planta edits that can be incorporated in the germline cells or inherited via seed. With the modification and adoption of various novel approaches currently being used in animal systems, it seems likely that non-transgenic genome editing will become routine in higher plants. 10.1007/s11248-021-00239-w
    Genome editing in cereal crops: an overview. Matres Jerlie Mhay,Hilscher Julia,Datta Akash,Armario-Nájera Victoria,Baysal Can,He Wenshu,Huang Xin,Zhu Changfu,Valizadeh-Kamran Rana,Trijatmiko Kurniawan R,Capell Teresa,Christou Paul,Stoger Eva,Slamet-Loedin Inez H Transgenic research Genome-editing technologies offer unprecedented opportunities for crop improvement with superior precision and speed. This review presents an analysis of the current state of genome editing in the major cereal crops- rice, maize, wheat and barley. Genome editing has been used to achieve important agronomic and quality traits in cereals. These include adaptive traits to mitigate the effects of climate change, tolerance to biotic stresses, higher yields, more optimal plant architecture, improved grain quality and nutritional content, and safer products. Not all traits can be achieved through genome editing, and several technical and regulatory challenges need to be overcome for the technology to realize its full potential. Genome editing, however, has already revolutionized cereal crop improvement and is poised to shape future agricultural practices in conjunction with other breeding innovations. 10.1007/s11248-021-00259-6
    Advances in application of genome editing in tomato and recent development of genome editing technology. Xia Xuehan,Cheng Xinhua,Li Rui,Yao Juanni,Li Zhengguo,Cheng Yulin TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik Genome editing, a revolutionary technology in molecular biology and represented by the CRISPR/Cas9 system, has become widely used in plants for characterizing gene function and crop improvement. Tomato, serving as an excellent model plant for fruit biology research and making a substantial nutritional contribution to the human diet, is one of the most important applied plants for genome editing. Using CRISPR/Cas9-mediated targeted mutagenesis, the re-evaluation of tomato genes essential for fruit ripening highlights that several aspects of fruit ripening should be reconsidered. Genome editing has also been applied in tomato breeding for improving fruit yield and quality, increasing stress resistance, accelerating the domestication of wild tomato, and recently customizing tomato cultivars for urban agriculture. In addition, genome editing is continuously innovating, and several new genome editing systems such as the recent prime editing, a breakthrough in precise genome editing, have recently been applied in plants. In this review, these advances in application of genome editing in tomato and recent development of genome editing technology are summarized, and their leaving important enlightenment to plant research and precision plant breeding is also discussed. 10.1007/s00122-021-03874-3
    Present and future prospects for wheat improvement through genome editing and advanced technologies. Li Shaoya,Zhang Chen,Li Jingying,Yan Lei,Wang Ning,Xia Lanqin Plant communications Wheat (, 2 = 6 = 42, AABBDD) is one of the most important staple food crops in the world. Despite the fact that wheat production has significantly increased over the past decades, future wheat production will face unprecedented challenges from global climate change, increasing world population, and water shortages in arid and semi-arid lands. Furthermore, excessive applications of diverse fertilizers and pesticides are exacerbating environmental pollution and ecological deterioration. To ensure global food and ecosystem security, it is essential to enhance the resilience of wheat production while minimizing environmental pollution through the use of cutting-edge technologies. However, the hexaploid genome and gene redundancy complicate advances in genetic research and precision gene modifications for wheat improvement, thus impeding the breeding of elite wheat cultivars. In this review, we first introduce state-of-the-art genome-editing technologies in crop plants, especially wheat, for both functional genomics and genetic improvement. We then outline applications of other technologies, such as GWAS, high-throughput genotyping and phenotyping, speed breeding, and synthetic biology, in wheat. Finally, we discuss existing challenges in wheat genome editing and future prospects for precision gene modifications using advanced genome-editing technologies. We conclude that the combination of genome editing and other molecular breeding strategies will greatly facilitate genetic improvement of wheat for sustainable global production. 10.1016/j.xplc.2021.100211
    Challenges and Opportunities for Clustered Regularly Interspaced Short Palindromic Repeats Based Molecular Biosensing. Bao Mengdi,Chen Qun,Xu Zhiheng,Jensen Erik C,Liu Changyue,Waitkus Jacob T,Yuan Xi,He Qian,Qin Peiwu,Du Ke ACS sensors Clustered regularly interspaced short palindromic repeats, CRISPR, has recently emerged as a powerful molecular biosensing tool for nucleic acids and other biomarkers due to its unique properties such as collateral cleavage nature, room temperature reaction conditions, and high target-recognition specificity. Numerous platforms have been developed to leverage the CRISPR assay for ultrasensitive biosensing applications. However, to be considered as a new gold standard, several key challenges for CRISPR molecular biosensing must be addressed. In this paper, we briefly review the history of biosensors, followed by the current status of nucleic acid-based detection methods. We then discuss the current challenges pertaining to CRISPR-based nucleic acid detection, followed by the recent breakthroughs addressing these challenges. We focus upon future advancements required to enable rapid, simple, sensitive, specific, multiplexed, amplification-free, and shelf-stable CRISPR-based molecular biosensors. 10.1021/acssensors.1c00530
    Engineering healthy crops: molecular strategies for enhancing the plant immune system. Frailie Tyler B,Innes Roger W Current opinion in biotechnology Crop diseases caused by viruses, bacteria, fungi, oomycetes and nematodes constitute major costs for farmers in terms of control measures and yield losses. Enhancing resistance to these pathogens via genetic modification or genome editing represents an economically and environmentally attractive path forward. Recent advances in our understanding of how plants detect pathogens and activate immune responses is now enabling enhancement of disease resistance traits. In particular, the recent determination of structures of both cell surface and intracellular immune receptors in plants in their activated states is providing new insights into how recognition complexes can be modified to expand recognition specificities to confer resistance to otherwise virulent pathogens. By expanding the repertoire of both cell surface and intracellular recognition systems, and combining them, it is expected that resistance to numerous diseases will be enhanced and will be more durable. 10.1016/j.copbio.2021.04.006
    CRISPR screens in plants: approaches, guidelines, and future prospects. The Plant cell Clustered regularly interspaced short palindromic repeat (CRISPR)-associated systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems have resulted in the development of powerful new screens to test gene functions at the genomic scale. While there is tremendous potential to map and interrogate gene regulatory networks at unprecedented speed and scale using CRISPR screens, their implementation in plants remains in its infancy. Here we discuss the general concepts, tools, and workflows for establishing CRISPR screens in plants and analyze the handful of recent reports describing the use of this strategy to generate mutant knockout collections or to diversify DNA sequences. In addition, we provide insight into how to design CRISPR knockout screens in plants given the current challenges and limitations and examine multiple design options. Finally, we discuss the unique multiplexing capabilities of CRISPR screens to investigate redundant gene functions in highly duplicated plant genomes. Combinatorial mutant screens have the potential to routinely generate higher-order mutant collections and facilitate the characterization of gene networks. By integrating this approach with the numerous genomic profiles that have been generated over the past two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will lead to great advances in functional and synthetic biology. 10.1093/plcell/koab099
    Getting back to the grass roots: harnessing specialized metabolites for improved crop stress resilience. Ding Yezhang,Northen Trent R,Khalil Ahmed,Huffaker Alisa,Schmelz Eric A Current opinion in biotechnology Roots remain an understudied site of complex and important biological interactions mediating plant productivity. In grain and bioenergy crops, grass root specialized metabolites (GRSM) are central to key interactions, yet our basic knowledge of the chemical language remains fragmentary. Continued improvements in plant genome assembly and metabolomics are enabling large-scale advances in the discovery of specialized metabolic pathways as a means of regulating root-biotic interactions. Metabolomics, transcript coexpression analyses, forward genetic studies, gene synthesis and heterologous expression assays drive efficient pathway discoveries. Functional genetic variants identified through genome wide analyses, targeted CRISPR/Cas9 approaches, and both native and non-native overexpression studies critically inform novel strategies for bioengineering metabolic pathways to improve plant traits. 10.1016/j.copbio.2021.05.010
    Applications and Major Achievements of Genome Editing in Vegetable Crops: A Review. Kim Young-Cheon,Kang Yeeun,Yang Eun-Young,Cho Myeong-Cheoul,Schafleitner Roland,Lee Jeong Hwan,Jang Seonghoe Frontiers in plant science The emergence of genome-editing technology has allowed manipulation of DNA sequences in genomes to precisely remove or replace specific sequences in organisms resulting in targeted mutations. In plants, genome editing is an attractive method to alter gene functions to generate improved crop varieties. Genome editing is thought to be simple to use and has a lower risk of off-target effects compared to classical mutation breeding. Furthermore, genome-editing technology tools can also be applied directly to crops that contain complex genomes and/or are not easily bred using traditional methods. Currently, highly versatile genome-editing tools for precise and predictable editing of almost any locus in the plant genome make it possible to extend the range of application, including functional genomics research and molecular crop breeding. Vegetables are essential nutrient sources for humans and provide vitamins, minerals, and fiber to diets, thereby contributing to human health. In this review, we provide an overview of the brief history of genome-editing technologies and the components of genome-editing tool boxes, and illustrate basic modes of operation in representative systems. We describe the current and potential practical application of genome editing for the development of improved nutritious vegetables and present several case studies demonstrating the potential of the technology. Finally, we highlight future directions and challenges in applying genome-editing systems to vegetable crops for research and product development. 10.3389/fpls.2021.688980
    Engineering Cas9 for human genome editing. Slaymaker Ian M,Gaudelli Nicole M Current opinion in structural biology Since the initial reports describing CRISPR-Cas9, labs across the globe have leveraged this valuable gene editing tool to alter the genomes of living cells. With the goal of generating more precise and efficient genome changes, scientists and engineers have mutated, evolved, and covalently altered Cas9 in order to predictably edit the genetic code. Here, we highlight recent advancements and contributions to the growing field of Cas9 engineering. We present key aspects of Cas9 engineering efforts focused on sgRNA manipulation, PAM-recognition, specificity, deaminase fusions, reverse-transcriptase fusions, and structural rearrangements of this important gene-modifying tool. 10.1016/j.sbi.2021.03.004
    CRISPR/Cas9-mediated genome editing: From basic research to translational medicine. Jacinto Filipe V,Link Wolfgang,Ferreira Bibiana I Journal of cellular and molecular medicine The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients. 10.1111/jcmm.14916
    Current progress and challenges in crop genetic transformation. Anjanappa Ravi B,Gruissem Wilhelm Journal of plant physiology Plant transformation remains the most sought-after technology for functional genomics and crop genetic improvement, especially for introducing specific new traits and to modify or recombine already existing traits. Along with many other agricultural technologies, the global production of genetically engineered crops has steadily grown since they were first introduced 25 years ago. Since the first transfer of DNA into plant cells using Agrobacterium tumefaciens, different transformation methods have enabled rapid advances in molecular breeding approaches to bring crop varieties with novel traits to the market that would be difficult or not possible to achieve with conventional breeding methods. Today, transformation to produce genetically engineered crops is the fastest and most widely adopted technology in agriculture. The rapidly increasing number of sequenced plant genomes and information from functional genomics data to understand gene function, together with novel gene cloning and tissue culture methods, is further accelerating crop improvement and trait development. These advances are welcome and needed to make crops more resilient to climate change and to secure their yield for feeding the increasing human population. Despite the success, transformation remains a bottleneck because many plant species and crop genotypes are recalcitrant to established tissue culture and regeneration conditions, or they show poor transformability. Improvements are possible using morphogenetic transcriptional regulators, but their broader applicability remains to be tested. Advances in genome editing techniques and direct, non-tissue culture-based transformation methods offer alternative approaches to enhance varietal development in other recalcitrant crops. Here, we review recent developments in plant transformation and regeneration, and discuss opportunities for new breeding technologies in agriculture. 10.1016/j.jplph.2021.153411
    CRISPR-Cas12a System for Biosensing and Gene Regulation. Shi Yuyan,Fu Xiaoyi,Yin Yao,Peng Fangqi,Yin Xia,Ke Guoliang,Zhang Xiaobing Chemistry, an Asian journal Clustered regularly interspaced short palindromic repeats (CRISPR) is a promising technology in the biological world. As one of the CRISPR-associated (Cas) proteins, Cas12a is an RNA-guided nuclease in the type V CRISPR-Cas system, which has been a robust tool for gene editing. In addition, due to the discovery of target-binding-induced indiscriminate single-stranded DNase activity of Cas12a, CRISPR-Cas12a also exhibits great promise in biosensing. This minireview not only gives a brief introduction to the mechanism of CRISPR-Cas12a but also highlights the recent developments and applications in biosensing and gene regulation. Finally, future prospects of the CRISPR-Cas12a system are also discussed. We expect this minireview will inspire innovative work on the CRISPR-Cas12a system by making full use of its features and advantages. 10.1002/asia.202100043
    Epigenetic editing: Dissecting chromatin function in context. Policarpi Cristina,Dabin Juliette,Hackett Jamie A BioEssays : news and reviews in molecular, cellular and developmental biology How epigenetic mechanisms regulate genome output and response to stimuli is a fundamental question in development and disease. Past decades have made tremendous progress in deciphering the regulatory relationships involved by correlating aggregated (epi)genomics profiles with global perturbations. However, the recent development of epigenetic editing technologies now enables researchers to move beyond inferred conclusions, towards explicit causal reasoning, through 'programing' precise chromatin perturbations in single cells. Here, we first discuss the major unresolved questions in the epigenetics field that can be addressed by programable epigenome editing, including the context-dependent function and memory of chromatin states. We then describe the epigenetic editing toolkit focusing on CRISPR-based technologies, and highlight its achievements, drawbacks and promise. Finally, we consider the potential future application of epigenetic editing to the study and treatment of specific disease conditions. 10.1002/bies.202000316
    Paving the way towards precise and safe CRISPR genome editing. Sledzinski Pawel,Dabrowska Magdalena,Nowaczyk Mateusz,Olejniczak Marta Biotechnology advances As the possibilities of CRISPR-Cas9 technology have been revealed, we have entered a new era of research aimed at increasing its specificity and safety. This stage of technology development is necessary not only for its wider application in the clinic but also in basic research to better control the process of genome editing. Research during the past eight years has identified some factors influencing editing outcomes and led to the development of highly specific endonucleases, modified guide RNAs and computational tools supporting experiments. More recently, large-scale experiments revealed a previously overlooked feature: Cas9 can generate reproducible mutation patterns. As a result, it has become apparent that Cas9-induced double-strand break (DSB) repair is nonrandom and can be predicted to some extent. Here, we review the present state of knowledge regarding the specificity and safety of CRISPR-Cas9 technology to define gRNA, protein and target-related problems and solutions. These issues include sequence-specific off-target effects, immune responses, genetic variation and chromatin accessibility. We present new insights into the role of DNA repair in genome editing and define factors influencing editing outcomes. In addition, we propose practical guidelines for increasing the specificity of editing and discuss novel perspectives in improvement of this technology. 10.1016/j.biotechadv.2021.107737
    Progression and application of CRISPR-Cas genomic editors. Yang Li,Tang Jing,Ma Xuelei,Lin Yuan,Ma Guorong,Shan Minghai,Wang Libin,Yang Yanhui Methods (San Diego, Calif.) Base editing technology is an efficient tool for genome editing, particularly in the correction of base mutations. Diverse base editing systems were developed according to the dCas9 or nCas9 linked with different deaminase or reverse transcriptase in the editors, including ABEs, CBEs, PEs and dual-functional of base editor (such as CGBE1, A&C-BEmax, ACBE, etc.). Currently, Base editing technology has been widely applied to various fields such as microorganisms, plants, animals and medicine for basic research and therapeutics. Here, we reviewed the advancement of base editing technology. We also discussed the application of base editors in different areas in the future. 10.1016/j.ymeth.2021.03.013
    CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement. Li Chao,Brant Eleanor,Budak Hikmet,Zhang Baohong Journal of Zhejiang University. Science. B Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations. 10.1631/jzus.B2100009
    Molecular Switch Engineering for Precise Genome Editing. Matsumoto Daisuke,Nomura Wataru Bioconjugate chemistry Genome editing technology commenced in 1996 with the discovery of the first zinc-finger nuclease. Application of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated protein 9 (Cas9) technology to genome editing of mammalian cells allowed researchers to use genome editing more easily and cost-effectively. However, one of the technological problems that remains to be solved is "off-target effects", which are unexpected mutations in nontarget DNA. One significant improvement in genome editing technology has been achieved with molecular/protein engineering. The key to this engineering is a "switch" to control function. In this review, we discuss recent efforts to design novel "switching" systems for precise editing using genome editing tools. 10.1021/acs.bioconjchem.1c00088
    Stressed Serratia curb CRISPR. Christen Beat Nature microbiology 10.1038/s41564-020-00848-x
    An introduction and use of the CRISPR-Cas systems. Singh Vijai Progress in molecular biology and translational science Clusters of regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) is a rapidly evolving field of targeted genome engineering. The type II CRISPR-Cas9 is used for genome editing of many organisms. Single guide RNA (sgRNA) can bind to Cas9 protein that can target desired sequences in presence of protospacer adjacent motif (PAM) sequences. This complex binds and generate a DSB that is repaired by NHEJ or HDR pathways, subsequently gene insertion/deletion (Indels) is generated that leads to change in the organism's genotype followed by its phenotype. In this chapter, CRISPR-mediated targeted genome editing in different lower organisms has been highlighted to promote its basic understanding to be applied for biotechnological, biomedical and therapeutic applications. 10.1016/bs.pmbts.2020.12.011
    History, evolution and classification of CRISPR-Cas associated systems. Agarwal Nisheeth,Gupta Radhika Progress in molecular biology and translational science This chapter provides a detailed description of the history of CRISPR-Cas and its evolution into one of the most efficient genome-editing strategies. The chapter begins by providing information on early findings that were critical in deciphering the role of CRISPR-Cas associated systems in prokaryotes. It then describes how CRISPR-Cas had been evolved into an efficient genome-editing strategy. In the subsequent section, latest developments in the genome-editing approaches based on CRISPR-Cas are discussed. The chapter ends with the recent classification and possible evolution of CRISPR-Cas systems. 10.1016/bs.pmbts.2020.12.012
    Advanced domestication: harnessing the precision of gene editing in crop breeding. Plant biotechnology journal Human population growth has increased the demand for food crops, animal feed, biofuel and biomaterials, all the while climate change is impacting environmental growth conditions. There is an urgent need to develop crop varieties which tolerate adverse growth conditions while requiring fewer inputs. Plant breeding is critical to global food security and, while it has benefited from modern technologies, it remains constrained by a lack of valuable genetic diversity, linkage drag, and an effective way to combine multiple favourable alleles for complex traits. CRISPR/Cas technology has transformed genome editing across biological systems and promises to transform agriculture with its high precision, ease of design, multiplexing ability and low cost. We discuss the integration of CRISPR/Cas-based gene editing into crop breeding to advance domestication and refine inbred crop varieties for various applications and growth environments. We highlight the use of CRISPR/Cas-based gene editing to fix desirable allelic variants, generate novel alleles, break deleterious genetic linkages, support pre-breeding and for introgression of favourable loci into elite lines. 10.1111/pbi.13576
    Can genetic engineering-based methods for gene function identification be eclipsed by genome editing in plants? A comparison of methodologies. Amritha P P,Shah Jasmine M Molecular genetics and genomics : MGG Finding and explaining the functions of genes in plants have promising applications in crop improvement and bioprospecting and hence, it is important to compare various techniques available for gene function identification in plants. Today, the most popular technology among researchers to identify the functions of genes is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based genome editing method. But by no means can we say that CRISPR/Cas9 is the go-to method for all purposes. It comes with its own baggage. Researchers will agree and have lived through at least seven more technologies deployed to find the functions of genes, which come under three umbrellas: 1. genetic engineering, 2. transient expression, and 3. chemical/physical mutagenesis. Each of the methods evolved when the previous one ran into an insurmountable problem. In this review, we compare the eight technologies against one another on 14 parameters. This review lays bare the pros and cons, and similarities and dissimilarities of various methods. Every method comes with its advantages and disadvantages. For example, the CRISPR/Cas9-based genome editing is an excellent method for modifying gene sequences, creating allelic versions of genes, thereby aiding the understanding of gene function. But it comes with the baggage of unwanted or off-target mutations. Then, we have methods based on random or targeted knockout of the gene, knockdown, and overexpression of the gene. Targeted disruption of genes is required for complete knockout of gene function, which may not be accomplished by editing. We have also discussed the strategies to overcome the shortcomings of the targeted gene-knockout and the CRISPR/Cas9-based methods. This review serves as a comprehensive guide towards the understanding and comparison of various technologies available for gene function identification in plants and hence, it will find application for crop improvement and bioprospecting related research. 10.1007/s00438-021-01769-y
    CRISPR-Cas12-based nucleic acids detection systems. Methods (San Diego, Calif.) Because of the outstanding contribution in genome editing, CRISPR has undoubtedly become the most popular technology around the world and two pioneers are awarded the Nobel Prize in Chemistry this year. Besides, along with the discovery of nonspecific trans-cleavage activities of several Cas proteins such as Cas12 and Cas13, many CRISPR-based molecular diagnostic systems have been successfully created, showing advantages in sensitivity, specificity and operation convenience. Among them, systems with Cas12, which targets DNA and trans-cleaves single-stranded DNA probes, are both simple and highly efficient. Here in this review, we mainly focus on the Cas12-based methods and briefly discuss their applications in nucleic acids detection and beyond. 10.1016/j.ymeth.2021.02.018
    Plant synthetic biology for producing potent phyto-antimicrobials to combat antimicrobial resistance. Tiwari Pragya,Khare Tushar,Shriram Varsha,Bae Hanhong,Kumar Vinay Biotechnology advances Inappropriate and injudicious use of antimicrobial drugs in human health, hygiene, agriculture, animal husbandry and food industries has contributed significantly to rapid emergence and persistence of antimicrobial resistance (AMR), one of the serious global public health threats. The crisis of AMR versus slower discovery of newer antibiotics put forth a daunting task to control these drug-resistant superbugs. Several phyto-antimicrobials have been identified in recent years with direct-killing (bactericidal) and/or drug-resistance reversal (re-sensitization of AMR phenotypes) potencies. Phyto-antimicrobials may hold the key in combating AMR owing to their abilities to target major microbial drug-resistance determinants including cell membrane, drug-efflux pumps, cell communication and biofilms. However, limited distribution, low intracellular concentrations, eco-geographical variations, beside other considerations like dynamic environments, climate change and over-exploitation of plant-resources are major blockades in full potential exploration phyto-antimicrobials. Synthetic biology (SynBio) strategies integrating metabolic engineering, RNA-interference, genome editing/engineering and/or systems biology approaches using plant chassis (as engineerable platforms) offer prospective tools for production of phyto-antimicrobials. With expanding SynBio toolkit, successful attempts towards introduction of entire gene cluster, reconstituting the metabolic pathway or transferring an entire metabolic (or synthetic) pathway into heterologous plant systems highlight the potential of this field. Through this perspective review, we are presenting herein the current situation and options for addressing AMR, emphasizing on the significance of phyto-antimicrobials in this apparently post-antibiotic era, and effective use of plant chassis for phyto-antimicrobial production at industrial scales along with major SynBio tools and useful databases. Current knowledge, recent success stories, associated challenges and prospects of translational success are also discussed. 10.1016/j.biotechadv.2021.107729
    Genome engineering for crop improvement and future agriculture. Gao Caixia Cell Feeding the ever-growing population is a major challenge, especially in light of rapidly changing climate conditions. Genome editing is set to revolutionize plant breeding and could help secure the global food supply. Here, I review the development and application of genome editing tools in plants while highlighting newly developed techniques. I describe new plant breeding strategies based on genome editing and discuss their impact on crop production, with an emphasis on recent advancements in genome editing-based plant improvements that could not be achieved by conventional breeding. I also discuss challenges facing genome editing that must be overcome before realizing the full potential of this technology toward future crops and food production. 10.1016/j.cell.2021.01.005
    State-of-the-Art in CRISPR Technology and Engineering Drought, Salinity, and Thermo-tolerant crop plants. Plant cell reports KEY MESSAGE:Our review has described principles and functional importance of CRISPR-Cas9 with emphasis on the recent advancements, such as CRISPR-Cpf1, base editing (BE), prime editing (PE), epigenome editing, tissue-specific (CRISPR-TSKO), and inducible genome editing and their potential applications in generating stress-tolerant plants. Improved agricultural practices and enhanced food crop production using innovative crop breeding technology is essential for increasing access to nutritious foods across the planet. The crop plants play a pivotal role in energy and nutrient supply to humans. The abiotic stress factors, such as drought, heat, and salinity cause a substantial yield loss in crop plants and threaten food security. The most sustainable and eco-friendly way to overcome these challenges are the breeding of crop cultivars with improved tolerance against abiotic stress factors. The conventional plant breeding methods have been highly successful in developing abiotic stress-tolerant crop varieties, but usually cumbersome and time-consuming. Alternatively, the CRISPR/Cas genome editing has emerged as a revolutionary tool for making efficient and precise genetic manipulations in plant genomes. Here, we provide a comprehensive review of the CRISPR/Cas genome editing (GE) technology with an emphasis on recent advances in the plant genome editing, including base editing (BE), prime editing (PE), epigenome editing, tissue-specific (CRISPR-TSKO), and inducible genome editing (CRISPR-IGE), which can be used for obtaining cultivars with enhanced tolerance to various abiotic stress factors. We also describe tissue culture-free, DNA-free GE technology, and some of the CRISPR-based tools that can be modified for their use in crop plants. 10.1007/s00299-021-02681-w
    CRISPR/Cas9-Mediated Gene Editing Revolutionizes the Improvement of Horticulture Food Crops. Wang Tian,Zhang Chunjiao,Zhang Hongyan,Zhu Hongliang Journal of agricultural and food chemistry Horticultural food crops are important sources of nutrients for humans. With the increase of the global population, enhanced horticulture food crop production has become a new challenge, and enriching their nutritional content has also been required. Gene editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), have accelerated crop improvement through the modification of targeted genomes precisely. Here, we review the development of various gene editors and compare their advantages and shortcomings, especially the newly emerging CRISPR/Cas systems, such as base editing and prime editing. We also summarize their practical applications in crop trait improvement, including yield, nutritional quality, and other consumer traits. 10.1021/acs.jafc.1c00104
    CRISPR/dCas-mediated transcriptional and epigenetic regulation in plants. Pan Changtian,Sretenovic Simon,Qi Yiping Current opinion in plant biology The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR Associated) system-mediated precise genome editing has revolutionized genome engineering due to ease of use and versatility of multiplexing. Catalytically inactivated Cas variants (dCas) further expand the usefulness of the CRISPR/Cas system for genetics studies and translational research without inducing DNA double-strand breaks. Fusion of diverse effector domains to dCas proteins empowers the CRISPR/dCas system as a multifunctional platform for gene expression regulation, epigenetic regulation and sequence-specific imaging. In this short review, we summarize the recent advances of CRISPR/dCas-mediated transcriptional activation and repression, and epigenetic modifications. We also highlight the future directions and broader applications of the CRISPR/dCas systems in plants. 10.1016/j.pbi.2020.101980
    Nanotechnology to advance CRISPR-Cas genetic engineering of plants. Demirer Gozde S,Silva Tallyta N,Jackson Christopher T,Thomas Jason B,W Ehrhardt David,Rhee Seung Y,Mortimer Jenny C,Landry Markita P Nature nanotechnology CRISPR-Cas genetic engineering of plants holds tremendous potential for providing food security, battling biotic and abiotic crop stresses caused by climate change, and for environmental remediation and sustainability. Since the discovery of CRISPR-Cas technology, its usefulness has been demonstrated widely, including for genome editing in plants. Despite the revolutionary nature of genome-editing tools and the notable progress that these tools have enabled in plant genetic engineering, there remain many challenges for CRISPR applications in plant biotechnology. Nanomaterials could address some of the most critical challenges of CRISPR genome editing in plants through improvements in cargo delivery, species independence, germline transformation and gene editing efficiency. This Perspective identifies major barriers preventing CRISPR-mediated plant genetic engineering from reaching its full potential, and discusses ways that nanoparticle technologies can lower or eliminate these barriers. We also describe advances that are needed in nanotechnology to facilitate and accelerate plant genome editing. Timely advancement of the application of CRISPR technologies in plant engineering is crucial for our ability to feed and sustain the growing human population under a changing global climate. 10.1038/s41565-021-00854-y
    Controlling and enhancing CRISPR systems. Shivram Haridha,Cress Brady F,Knott Gavin J,Doudna Jennifer A Nature chemical biology Many bacterial and archaeal organisms use clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) systems to defend themselves from mobile genetic elements. These CRISPR-Cas systems are classified into six types based on their composition and mechanism. CRISPR-Cas enzymes are widely used for genome editing and offer immense therapeutic opportunity to treat genetic diseases. To realize their full potential, it is important to control the timing, duration, efficiency and specificity of CRISPR-Cas enzyme activities. In this Review we discuss the mechanisms of natural CRISPR-Cas regulatory biomolecules and engineering strategies that enhance or inhibit CRISPR-Cas immunity by altering enzyme function. We also discuss the potential applications of these CRISPR regulators and highlight unanswered questions about their evolution and purpose in nature. 10.1038/s41589-020-00700-7
    Plant genome editing: ever more precise and wide reaching. Sukegawa Satoru,Saika Hiroaki,Toki Seiichi The Plant journal : for cell and molecular biology Genome-editing technologies consisting of targeted mutagenesis and gene targeting enable us to modify genes of interest rapidly and precisely. The discovery in 2012 of CRISPR/Cas9 systems and their development as sequence-specific nucleases has brought about a paradigm shift in biology. Initially, CRISPR/Cas9 was applied in targeted mutagenesis to knock out a target gene. Thereafter, advances in genome-editing technologies using CRISPR/Cas9 developed rapidly, with base editing systems for transition substitution using a combination of Cas9 nickase and either cytidine or adenosine deaminase being reported in 2016 and 2017, respectively, and later in 2021 bringing reports of transversion substitution using Cas9 nickase, cytidine deaminase and uracil DNA glycosylase. Moreover, technologies for gene targeting and prime editing systems using DNA or RNA as donors have also been developed in recent years. Besides these precise genome-editing strategies, reports of successful chromosome engineering using CRISPR/Cas9 have been published recently. The application of genome editing to crop breeding has advanced in parallel with the development of these technologies. Genome-editing enzymes can be introduced into plant cells, and there are now many examples of crop breeding using genome-editing technologies. At present, it is no exaggeration to say that we are now in a position to be able to modify a gene precisely and rearrange genomes and chromosomes in a predicted way. In this review, we introduce and discuss recent highlights in the field of precise gene editing, chromosome engineering and genome engineering technology in plants. 10.1111/tpj.15233
    Applying gene editing to tailor precise genetic modifications in plants. Van Eck Joyce The Journal of biological chemistry The ability to tailor alterations in genomes, including plant genomes, in a site-specific manner has been greatly advanced through approaches that reduced the complexity and time of genome sequencing along with development of gene editing technologies. These technologies provide a valuable foundation for studies of gene function, metabolic engineering, and trait modification for crop improvement. Development of genome editing methodologies began ∼20 years ago, first with meganucleases and followed by zinc finger nucleases, transcriptional activator-like effector nucleases and, most recently, clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein (Cas) (CRISPR/Cas), which is by far the most utilized method. The premise of CRISPR/Cas centers on the cleaving of one or both DNA strands by a Cas protein, an endonuclease, followed by mending of the DNA by repair mechanisms inherent in cells. Its user-friendly construct design, greater flexibility in targeting genomic regions, and cost-effective attributes have resulted in it being widely adopted and revolutionizing precise modification of the genomes of many organisms. Indeed, the CRISPR/Cas system has been utilized for gene editing in many plant species, including important food crops, such as maize, wheat, rice, and potatoes. This review summarizes the various approaches, including the most recent designs being used to make modifications from as small as a single-base-pair change to insertion of DNA fragments. On the gene expression level, strategies are presented that make it possible to knock out or modulate through activation and repression. Also discussed are prerequisites necessary for CRISPR/Cas-mediated editing as well as the current challenges. 10.1074/jbc.REV120.010850