Central Dogma of Molecular Biology

DNA cryptography is an emerging field of DNA computing research in information security world. Still this field is in theoretical stage and not in active practice. To overcome this lacuna the present study proposed a fast and secured hybrid algorithm using DES (Data Encryption Standard) with biological concept CDMB (Central Dogma of Molecular biology) developed and implemented in high level programming language. This edifice has enabled to do computations digitally and gave the high level of security, effectiveness and applicability.

Plant diseases are still affecting the world food security by lowering the production of large crops. In this review, the precision immunity concept, which involves the specific editing of endogenous plant genes to boost innate immunity, is critically assessed as the use of CRISPR/Cas-mediated genome editing to increase disease resistance. Authors reviewed the current literature on disruption of susceptibility (S) gene, control of immune signaling pathways, and allele-engineering in crops, including rice, wheat, and tomato. The review also covers the developments in multiplex editing, base editing, prime editing, and the unique functions of the system based on Cas12 (DNA targeting) and Cas13 (RNA targeting) in controlling DNA-and RNA-based pathogenesis. With respect to the editing outcomes, a few brief remarks are made on mechanistic aspects of repair pathways such as non-homologous end joining and homology-directed repair. Technical issues, regulatory changes, and biosafety issues are also evaluated. Altogether, as the existing evidence indicates, CRISPR-based approaches possess enormous capacity to aid in the creation of longlasting and climate-resilient disease resistance in crops.

Plant diseases are still affecting the world food security by lowering the production of large crops. In this review, the precision immunity concept, which involves the specific editing of endogenous plant genes to boost innate immunity, is critically assessed as the use of CRISPR/Cas-mediated genome editing to increase disease resistance. Authors reviewed the current literature on disruption of susceptibility (S) gene, control of immune signaling pathways, and allele-engineering in crops, including rice, wheat, and tomato. The review also covers the developments in multiplex editing, base editing, prime editing, and the unique functions of the system based on Cas12 (DNA targeting) and Cas13 (RNA targeting) in controlling DNA- and RNA-based pathogenesis. With respect to the editing outcomes, a few brief remarks are made on mechanistic aspects of repair pathways such as non-homologous end joining and homology-directed repair. Technical issues, regulatory changes, and biosafety issues are also evaluated. Altogether, as the existing evidence indicates, CRISPR-based approaches possess enormous capacity to aid in the creation of long-lasting and climate-resilient disease resistance in crops.

Genome editing has been well recognized as a genome engineering tool that allows scientists to permanently modify the DNA contented at a particular genomic location. Earlier, it was carried out by delivering a DNA template with a long homologous arm to the targeted genomic site. This process was time-consuming and required synthesis and the delivery of a long DNA template. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 are three extensively used genome editing technologies that were developed in response to these limitations. This paper provides a comparative analysis of the origin, structure, function, and working advantages over each other, limitations, and their application in different organisms for disease treatment and genetic modifications of these three technologies. ZFNs are earliest genome editing technology. ZFN is a protein having Zn finger domains that recognize the DNA and the Fok domain that cuts the DNA sequences. TALENs are similar to ZFNs but use transcription activator-like effectors instead of zinc fingers to recognize DNA. CRISPR-Cas9 is more recent technology that uses RNA guides to target specific DNA sequences. One advantage of ZFNs and TALENs is their high specificity but they are time-consuming and expensive processes to design and synthesize each nuclease for a specific target sequence. Whereas CRISPR-Cas9 is faster and more cost-effective, as it requires only simple RNA guide design to target specific DNA sequence. In conclusion, ZFNs, TALENs, and CRISPR-Cas9 are all useful genome editing technologies. Each of these technologies have advantages and limitations that will be discussed in the article.

At the core of all living cells lies DNA, RNA, and protein - a deeply conserved communication architecture encompassing transcription, translation, the genetic code and the Central Dogma. No existing theory explains why this specific DNA-RNA-protein system exists or why it exhibits its particular structure. Here, communication is analysed in a substrate-independent regime, free of space, time, and matter, under logical constraints alone. A unique triadic, unidirectional architecture emerges: the Non-Physical Communication Architecture (NPCA). The NPCA maps directly to the DNA-RNA-protein system, thereby providing a unified account of the DNA-RNA-protein information flow as embodied logic rather than historical accidents of prebiotic chemistry. Life is defined as the embodied NPCA (E-NPCA) in matter. This framework resolves puzzles in viral classification, cryptobiosis and homochirality, while implying a rare, threshold origin of life with consequences for the Fermi Paradox.

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The CRISPR-Cas system has rapidly reached a huge popularity as a new, powerful method for precise DNA editing and genome reengineering. In Synthetic Biology, the CRISPR-Cas type II system has inspired the construction of a novel class of RNA-based transcription factors. In their simplest form, they are made of a CRISPR RNA molecule, which targets a promoter sequence, and a deficient Cas9 (i.e. deprived of any nuclease activity) that has been fused to an activation or a repression domain. Up-and downregulation of single genes in mammalian and yeast cells have been achieved with satisfactory results. Moreover, the construction of CRISPR-based transcription factors is much simpler than the assembly of synthetic proteins such as the Transcription Activator-Like effectors. However, the feasibility of complex synthetic networks fully based on the CRISPR-dCas9 technology has still to be proved and new designs, which take into account different CRISPR types, shall be investigated.

DNA cryptography is an emerging field of DNA computing research in information security world. Still this field is in theoretical stage and not in active practice. To overcome this lacuna the present study proposed a fast and secured hybrid algorithm using DES (Data Encryption Standard) with biological concept CDMB (Central Dogma of Molecular biology) developed and implemented in high level programming language. This edifice has enabled to do computations digitally and gave the high level of security, effectiveness and applicability.

We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference collaborate to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that prevented translation of the main ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2. We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly upregulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs. Gene expression regulation enables differential decoding of identical genetic material. It has generally been studied as a set of sequential processes, with transcription setting the core pattern of expression and translational and post-translational regulation modulating the final output. The concept of an exclusively linear model for the regulation of genetic information decoding is partly the result of the largely isolated discovery and subsequent study of each regulatory step. This approach has been necessary for and successful in providing a deep understanding of the biochemical mechanisms that mediate gene regulation, as well as for defining the types of regulatory mechanisms that exist at each level. Such regulatory mechanisms include, for example, transcriptional interference, in which transcription from one promoter locally represses transcription from another (Hausler and Somerville 1979; Adhya and

Living cells and organisms are complex physical systems. Does their organization or complexity primarily rely on the intra-molecular crystalline structure of genetic nucleic acid sequences? Or is it, as critics of the 'gene-centred' perspective claim, predominantly a result of the inter-and supra-molecular -thus 'holistic' -network dynamics of genetic and various extra-genetic factors? The twentieth-century successes in several branches of genetics caused intensive focus on the causal role of genes in the biochemistry, development and evolution of living organisms, resulting in a relative abstraction or even neglect of life's complex systems dynamics. Today, however, partly due to the success of systems biology, a number of authors defend life's systems complexity while criticizing the gene-centred approach. Here, we offer a way out of the impasse of the gene-centred 'versus' complex systems perspective to arrive at a more balanced and complete understanding of life's multifaceted nature. After sketching the conceptual and historical background of the controversy, we show how the present state of knowledge in biology vindicates both the holistically complex and gene-centred nature of life on Earth, but decisively falsifies extreme genetic 'determinism' and 'reductionism' as well as extreme 'gene-de-centrism'. Contrary to what is often claimed, the fact that genes are one among many extra-genetic causal factors contributing to the biochemistry and development of cells and organisms, only undermines or falsifies genetic determinism and reductionism but not necessarily gene-centrism. Some implications for evolutionary theory, i.e., for the controversy between the Modern Synthesis and an 'Extended Synthesis', are outlined.

Floriculture is an integral part of modern agriculture. Genetic transformation techniques pave way for the development of  biotechnology assisted novel varieties which are appealing to customers. Customer preference in floriculture changes rapidly with time. Recently discovered CRISPR/Cas genome editing technique is useful in editing key genes attributing to a trait with the enhanced aesthetic appeal of plants. This chapter primarily discusses the application of biotechnology-based tools for improvement of floriculture plants to keep up with consumer appeal

Cold water, cold weather and cold climate are expanding problems of rice growing areas. Climate change enhances more extreme events and unfavorable environment in certain areas for rice growers. Cold stress affects rice growth and yield and ultimately, limits rice productivity. The complex mechanisms affecting the molecular and physiological changes that enable adaptive response to cold stress in rice. In this complex mechanism, the cell perceives cold stress, signal transduction through the cell memebrance or channels to nucleus or chromosomes which response as transcriptional activated or deactivated genes using gene expression pathways. Multifunctional (genetics, molecular, physiological and physical) analysis and approaches solve these complex traits (cold stress) related problems. With the objective of this study was to generate and develop diverse mutants (lines/varieties) for cold tolerance on rice, we determined the possible target sites and its relation with proteins by using OPT8511 cold sensitive) sequence. About 172 proteins are affected by this gene editing system and among them, 61 proteins were found as direct or indirect relation and association with cold responsive traits or enzymes related to nine chromosomes. The collection of proteins, their position and their functions has given a good command in the future study in relation of cold response of rice and other crops, and also have new possibilities of diversity for rice breeding in future and farmers will be benefited in double or triple rice growing zones and cold sensitive areas..

Rice (Oryza sativa) is one of the most important staple food crops feeding more than half of the world’s population. One of the requirements for future sustainable rice production is to develop drought tolerant varieties. We have identified a number of drought sensitive tagged lines by screening our rice activation tagging population. Two of the sensitive lines, AH13391 and AH17392, exhibit reduced drought tolerance compared to the controls, and have a single T-DNA in a region next to an ATPase-associated with diverse cellular activities (AAA)-like gene, respectively. Constitutive overexpression of either AAA-like gene (OsAAA-1 and OsAAA-2) significantly reduced the drought tolerance, whereas knocking them out by CRISPR-Cas9 significantly increased grain yield under both drought and well-watered field conditions. Comparative analysis of different OsAAA-1-edited variations shows that the core AAA ATPase domain and the C-terminal end of OsAAA-1 protein are important for its function i...

This study explores the potential of harnessing epigenetic modifications for targeted trait manipulation in Pakistani wheat varieties. Epigenetic regulation is pivotal in modulating gene expression patterns and can be leveraged to enhance agronomic traits. A diverse panel of Pakistani wheat varieties was subjected to comprehensive epigenetic profiling, encompassing DNA methylation and histone modification analyses. CRISPR-based epigenetic editing targeted specific genes associated with disease resistance, yield, and nutritional content. Field trials were conducted to evaluate the agronomic performance of edited lines. Epigenetic profiling revealed distinct DNA methylation and histone modification patterns across the selected wheat varieties. Targeted epigenetic editing amplified disease resistance, enhanced yield potential, and enriched nutritional content. Disease incidence and severity were significantly reduced in edited lines. Additionally, increased tiller number, larger grain size, and elevated levels of essential nutrients were observed. This research demonstrates the feasibility and efficacy of employing epigenetic modifications for precise trait manipulation in Pakistani wheat. The study provides valuable insights into the regulatory mechanisms underlying disease resistance, yield, and nutritional traits. Collaborative efforts among geneticists, molecular biologists, and plant breeders are essential in further refining and implementing these innovative breeding techniques. The findings pave the way for developing resilient and nutritionally enriched wheat varieties, contributing to global food security and agricultural sustainability.

Background Epigenome editing refers to the targeted reprogramming of genomic loci using an EpiEditor which may consist of dCas9, DNMT3A/3L and sgRNA. Methylation of the locus can lead to a modulation of gene expression. Allele-specific DNA methylation (ASM) refers to the targeted methylation delivery only to one allele of a locus. In the context of diseases caused by a dominant mutation, the selective DNA methylation of the mutant allele could be used to repress its expression but retain the functionality of the normal gene. Results To set up allele-specific targeted DNA methylation, target regions were selected from hypomethylated CGIs bearing a SNP in their promoters in the HEK293 cell line. We aimed at delivering maximum DNA methylation with highest allelic specificity in the targeted regions. Placing SNPs in the PAM or seed regions of the sgRNA, we designed 24 different sgRNAs targeting single alleles. We achieved efficient ASM in multiple cases, such as ISG15, MSH6, GPD1L, MRPL...

Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made with respect to CRISPR-based multiplex genome editing of industrial bacteria and eukaryotic microorganisms. We also review the state of the art in terms of gene expression regulation using CRISPRi and CRISPRa. Finally, we summarize the pillars for efficient multiplexed genome editing and present our view on future developments and applications of CRISPR-Cas tools for multiplex genome editing.

DNA cryptography is a technology of bio science to encrypt large message in compact volume. Now a day, researchers are going to research in the field of secure data transmission. Hiding the encrypted message is important part of Cryptography. Hidden message is in the form of DNA sequence, image, audio and video, which is used to prevent important data from the intruders. In this paper, a new cryptography technique is proposed using Symmetric Key Exchange, one-time pad scheme and DNA hybridization to minimize time complexity.XOR operation with OTP DNA sequence is used as encryption technique based on DNA cryptography. Symmetric Key Exchange is presenting a secure key generation scheme. This method is very efficient in encrypting, hiding, transmitting and preventing powerful attacks.

DNA cryptography is a technology of bio science to encrypt large message in compact volume. Now a day, researchers are going to research in the field of secure data transmission. Hiding the encrypted message is important part of Cryptography. Hidden message is in the form of DNA sequence, image, audio and video, which is used to prevent important data from the intruders. In this paper, a new cryptography technique is proposed using Symmetric Key Exchange, one-time pad scheme and DNA hybridization to minimize time complexity.XOR operation with OTP DNA sequence is used as encryption technique based on DNA cryptography. Symmetric Key Exchange is presenting a secure key generation scheme. This method is very efficient in encrypting, hiding, transmitting and preventing powerful attacks.

Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants for the desired properties. However, DE methods have for a long time been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools that circumvent enduring barriers such as longer life cycle, small library sizes, and low mutation rates have been developed to facilitate DE in native genetic environments of multicellular organisms. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE schemes by enabling base substitutions and randomization of targeted DNA sequences. BE-TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on the recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements. [BMB Reports 2024; 57(1): 30-39]

Chromosomal inversions are recurrent rearrangements that occur between different plant isolates or cultivars. Such inversions may underlie reproductive isolation in evolution and represent a major obstacle for classical breeding as no crossovers can be observed between inverted sequences on homologous chromosomes. The heterochromatic knob (hk4S) on chromosome 4 is the most well-known inversion of Arabidopsis. If a knob carrying accession such as Col-0 is crossed with a knob-less accession such as Ler-1, crossovers cannot be recovered within the inverted region. Our work shows that by egg-cell specific expression of the Cas9 nuclease from Staphylococcus aureus, a targeted reversal of the 1.1 Mb long hk4Sinversion can be achieved. By crossing Col-0 harbouring the rearranged chromosome 4 with Ler-1, meiotic crossovers can be restored into a region with previously no detectable genetic exchange. The strategy of somatic chromosome engineering for breaking genetic linkage has huge potential for application in plant breeding.

CRISPR–Cas genome editing technology developed from prokaryotes has transformed the molecular biology of plants past all assumptions. CRISPR–Cas, which is distinguished by its resilience, relatively high specificity, and easy implementation, enables specific genetic modification of crops, allowing for the creation of germplasms with favorable characters and the development of innovative, highly efficient agricultural systems. Moreover, many new biotechnologies in the framework of CRISPR–Cas platforms have bolstered basic research as well as synthetic biology toolkit of plants. In this article, initially, we provide a brief overview of CRISPR–Cas gene editing, emphasis on the modern, most specific gene-editing techniques, such as prime and base editing. Following that, the major role of CRISPR–Cas in plants in enhancing pesticide and disease resistance, quality, yield, breeding, and faster domestication are next discussed. In this review, we discuss the current advancements in plant ...

The CRISPR system has become heavily utilized in biomedical research as a tool for genomic editing as well as for site-specific chromosomal localization of specific proteins. For example, we developed a CRISPR-based methodology for enriching a specific genomic locus of interest for proteomic analysis in Saccharomyces cerevisiae, which utilized a guide RNA-targeted, catalytically dead Cas9 (dCas9) as an affinity reagent. To more comprehensively evaluate the genomic specificity of using dCas9 as a site-specific tool for chromosomal studies, we performed dCas9-mediated locus enrichment followed by next-generation sequencing on a genome-wide scale. As a test locus, we used the ARS305 origin of replication on chromosome III in S. cerevisiae. We found that enrichment of this site is highly specific, with virtually no off-target enrichment of unique genomic sequences. The high specificity of genomic localization and enrichment suggests that dCas9-mediated technologies have promising potent...

Currently, poor biodiversity has raised challenges in the breeding and cultivation of tomatoes, which originated from the Andean region of Central America, under global climate change. Meanwhile, the wild relatives of cultivated tomatoes possess a rich source of genetic diversity but have not been extensively used for the genetic improvement of cultivated tomatoes due to the possible linkage drag of unwanted traits from their genetic backgrounds. With the advent of new plant breeding techniques (NPBTs), especially CRISPR/Cas-based genome engineering tools, the high-precision molecular breeding of tomato has become possible. Further, accelerated introgression or de novo domestication of novel and elite traits from/to the wild tomato relatives to/from the cultivated tomatoes, respectively, has emerged and has been enhanced with high-precision tools. In this review, we summarize recent progress in tomato precision genome editing and its applications for breeding, with a special focus on CRISPR/Cas-based approaches. Future insights and precision tomato breeding scenarios in the CRISPR/Cas era are also discussed.

The soybean is a valuable legume crop cultivated for its oil and protein which is used widely as food for humans and feed for livestock as well as in biofuel production. The genetic improvement of the soybean needs to be accelerated to boost its productivity and enhance its resilience to changing environments. In recent years, CRISPR/Cas9 has become a powerful and robust genome editing system for manipulating traits of various crop plants including soybean. This cutting-edge biotechnological tool has been extensively used as a means for improving crop quality and yields, disease-resistance, tolerance to adverse environmental conditions, and production of plant-based materials. This review presents a brief mechanism of the CRISPR/Cas9 system followed by its application in soybean improvement. It also highlights some prospects of using the CRISPR/Cas9 system in soybean research.

The soybean is a valuable legume crop cultivated for its oil and protein which is used widely as food for humans and feed for livestock as well as in biofuel production. The genetic improvement of the soybean needs to be accelerated to boost its productivity and enhance its resilience to changing environments. In recent years, CRISPR/Cas9 has become a powerful and robust genome editing system for manipulating traits of various crop plants including soybean. This cutting-edge biotechnological tool has been extensively used as a means for improving crop quality and yields, disease-resistance, tolerance to adverse environmental conditions, and production of plant-based materials. This review presents a brief mechanism of the CRISPR/Cas9 system followed by its application in soybean improvement. It also highlights some prospects of using the CRISPR/Cas9 system in soybean research.

Transgene-free genome editing of plants in the T0 generation is highly desirable but challenging, especially in perennials and vegetatively propagated plants. Here, we investigated the co-editing strategy for generating transgene-free, gene-edited plants viaAgrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFPin planta. Specifically, CBE/gRNA was used to base edit theALSgene to confer resistance to herbicide chlorsulfuron as a selection marker, which has no negative effects on plant phenotypes; Cas12a/crRNA was used for editing genes(s) of interest; GFP was used for selecting transgene-free transformants. Using this approach, transgene-free genome-edited plants were efficiently generated for various genes (either individual or multiplex) in tomato, tobacco, potato, and citrus in the T0 generation. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 8% to 50%. Whole genom...

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Cold water, cold weather and cold climate are expanding problems of rice growing areas. Climate change enhances more extreme events and unfavorable environment in certain areas for rice growers. Cold stress affects rice growth and yield and ultimately, limits rice productivity. The complex mechanisms affecting the molecular and physiological changes that enable adaptive response to cold stress in rice. In this complex mechanism, the cell perceives cold stress, signal transduction through the cell memebrance or channels to nucleus or chromosomes which response as transcriptional activated or deactivated genes using gene expression pathways. Multifunctional (genetics, molecular, physiological and physical) analysis and approaches solve these complex traits (cold stress) related problems. With the objective of this study was to generate and develop diverse mutants (lines/varieties) for cold tolerance on rice, we determined the possible target sites and its relation with proteins by using OPT8511 cold sensitive) sequence. About 172 proteins are affected by this gene editing system and among them, 61 proteins were found as direct or indirect relation and association with cold responsive traits or enzymes related to nine chromosomes. The collection of proteins, their position and their functions has given a good command in the future study in relation of cold response of rice and other crops, and also have new possibilities of diversity for rice breeding in future and farmers will be benefited in double or triple rice growing zones and cold sensitive areas..

Even though the accrual of transcripts is implicated in distinct disease states, our knowledge regarding their functional role remains obscure. The CRISPR system has surged at the forefront of genome engineering tools in the field of RNA modulation. In the present review, we discuss some exciting applications of the CRISPR system, including the manipulation of RNA sequences, the visualization of chromosomal loci in living cells and the modulation of transcription. The CRISPR system has been documented to be very reliable and specific in altering gene expression, via leveraging inactive catalytically dead CRISPR-associated protein 9 (Cas9). In the present review, the CRISPR system is presented as an eminent tool for the meticulous analysis of gene regulation, loci mapping and complex pathways.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system was initially discovered as an underlying mechanism for conferring adaptive immunity to bacteria and archaea against viruses. Over the past decade, this has been repurposed as a genome-editing tool. Numerous gene editing-based crop improvement technologies involving CRISPR/Cas platforms individually or in combination with next-generation sequencing methods have been developed that have revolutionized plant genome-editing methodologies. Initially, CRISPR/Cas nucleases replaced the earlier used sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), to address the problem of associated off-targets. The adaptation of this platform led to the development of concepts such as epigenome editing, base editing, and prime editing. Epigenome editing employed epi-effectors to manipulate chromatin structure, while base ed...

Mounting evidence has called into question our understanding of the role that the central dogma of molecular biology plays in human pathology. The conventional view that elucidating the mechanisms for translating genes into proteins can account for a panoply of diseases has proven incomplete. Landmark studies point to epigenetics as a missing piece of the puzzle. However, technological limitations have hindered the study of specific roles for histone post-translational modifications, DNA modifications, and non-coding RNAs in regulation of the epigenome and chromatin structure. This feature highlights CRISPR systems, including CRISPR-Cas9, as novel tools for targeted epigenome editing. It summarizes recent developments in the field, including integration of optogenetic and functional genomic approaches to explore new therapeutic opportunities, and underscores the importance of mitigating current limitations in the field. This comprehensive, analytical assessment identifies current re...

The year 2020 marks a decade since the first gene-edited plants were generated using homing endonucleases and zinc finger nucleases. The advent of CRISPR/Cas9 for gene-editing in 2012 was a major science breakthrough that revolutionized both basic and applied research in various organisms including plants and consequently honored with “The Nobel Prize in Chemistry, 2020.” CRISPR technology is a rapidly evolving field and multiple CRISPR-Cas derived reagents collectively offer a wide range of applications for gene-editing and beyond. While most of these technological advances are successfully adopted in plants to advance functional genomics research and development of innovative crops, others await optimization. One of the biggest bottlenecks in plant gene-editing has been the delivery of gene-editing reagents, since genetic transformation methods are only established in a limited number of species. Recently, alternative methods of delivering CRISPR reagents to plants are being explo...

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system was initially discovered as an underlying mechanism for conferring adaptive immunity to bacteria and archaea against viruses. Over the past decade, this has been repurposed as a genome-editing tool. Numerous gene editing-based crop improvement technologies involving CRISPR/Cas platforms individually or in combination with next-generation sequencing methods have been developed that have revolutionized plant genome-editing methodologies. Initially, CRISPR/Cas nucleases replaced the earlier used sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), to address the problem of associated off-targets. The adaptation of this platform led to the development of concepts such as epigenome editing, base editing, and prime editing. Epigenome editing employed epi-effectors to manipulate chromatin structure, while base ed...

The recent global climate change has directly impacted major biotic and abiotic stress factors affecting crop productivity worldwide. Therefore, the need of the hour is to develop sustainable multiple stress tolerant crops through modern biotechnological approaches to cope with climate change. Hybrid proline rich proteins (HyPRPs) are the cell-wall structural proteins, which contain an N-terminal repetitive proline-rich domain and a C-terminal conserved eight-cysteine motif domain. HyPRPs are known to regulate multiple abiotic and biotic stress responses in plants. Recently, a few HyPRPs have been characterized as negative regulators of abiotic and biotic stress responses in different plants. Disruption of such negative regulators for desirable positive phenotypic traits has been made possible through the advent of advanced genome engineering tools. In the past few years, CRISPR/ Cas9 has emerged as a novel breakthrough technology for crop improvement by target specific editing of known negative regulatory host genes. Here, we have described the mechanism of action and the role of known HyPRPs in regulating different biotic and abiotic stress responses in major crop plants. We have also discussed the importance of the CRISPR/Cas9 based genome editing system in targeting known negative regulatory HyPRPs for multi-stress crop tolerance using the tomato crop model. Application of genome editing to manipulate the HyPRPs of major crop plants holds promise in developing newer stress management methods in this rapidly changing climate and would lead in the future to sustain crop productivity.

Yeast is a single-celled and versatile microorganism that is widely used in various industries, such as brewing, food, and biotechnology. However, not all yeast strains possess the desired characteristics required for optimal production. Therefore, methods of yeast strain improvement have been developed to enhance the desirable traits of yeast strains and to benefit industries involving yeast. Yeast strains with desirable properties are crucial for these industries to achieve optimal productivity and quality.

Key message This review illustrates how far we have come since the emergence of GE technologies and how they could be applied to obtain superior and sustainable crop production. Abstract The main challenges of today’s agriculture are maintaining and raising productivity, reducing its negative impact on the environment, and adapting to climate change. Efficient plant breeding can generate elite varieties that will rapidly replace obsolete ones and address ongoing challenges in an efficient and sustainable manner. Site-specific genome editing in plants is a rapidly evolving field with tangible results. The technology is equipped with a powerful toolbox of molecular scissors to cut DNA at a pre-determined site with different efficiencies for designing an approach that best suits the objectives of each plant breeding strategy. Genome editing (GE) not only revolutionizes plant biology, but provides the means to solve challenges related to plant architecture, food security, nutrient conte...

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome editing approaches to develop improved crops. Often considered genome ‘parasites’, transposable elements (TEs) evolved to insert their DNA seamlessly into genomes. TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type. We developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the TE’s natural ability to precisely insert into the genome. Inspired by CRISPR-associated transposases (CASTs) that target transposition in a programmable manner in bacteria, we generated a synthetic CAST by fusing the rice Pong TE transposase protein to the Cas9 or CPF1 programmable nucleases. We demonstrated sequence-specific targeted delivery (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and gene expression cassette ...

Every year, the world's population grows, and to sustain this enormous population, food consumption must likewise massively double. However, because of unpredictable environmental fluctuations, plants are unable to produce the necessary amount of food, and several variables have negatively impacted their growth. Abiotic stresses like drought and salinity stress have already caused too much destruction in crop yield and are expected to decrease the yield in coming years. Also, biotic factors cause a severe threat to plant production. Conventional strategies are not much effective in controlling these disasters in plants. Biotechnological innovations have much potential to modify the tolerance mechanism in plants or engineer another process to make tolerant varieties. CRISPR/Cas technology has emerged as a promising single or multiple-site-directed mutagenesis tool. The CRISPR Cas technique alters the key genes that code for the enzymes that support metabolic pathways. The end goal is to raise fruit quality. It has advantages compared to conventional random mutagenesis methods due to its high efficacy, precision, and low risks of off-target activities. In the present study, CRISPR/Cas technology potential has been described as modifying plant genomes for high yields under various biotic/abiotic stresses.

Genome editing provides a new therapeutic strategy to cure genetic diseases. The recently developed CRISPR-Cas9 base editing technology has shown great potential to repair the majority of pathogenic point mutations in the patient's DNA precisely. Base editor is the fusion of a Cas9 nickase with a base-modifying enzyme that can change a nucleotide on a single strand of DNA without generating double-stranded DNA breaks. However, a major limitation in applying such a system is the prerequisite of a protospacer adjacent motif sequence at the desired position relative to the target site. Progress has been made to increase the targeting scope of base editors by engineering SpCas9 protein variants, establishing systems with broadened editing windows, characterizing new SpCas9 orthologs, and developing prime editing technology. In this review, we discuss recent progress in the development of CRISPR base editing, focusing on its targeting scope, and we provide a workflow for selecting a suitable base editor based on the target nucleotide sequences.

Rice (Oryza sativa) is an important staple food crop worldwide; to meet the growing nutritional requirements of the increasing population in the face of climate change, qualitative and quantitative traits of rice need to be improved. Stress-tolerant crop varieties must be developed with stable or higher yields under stress conditions. Genome editing and speed breeding have improved the accuracy and pace of rice breeding. New breeding technologies including genome editing have been established in rice, expanding the potential for crop improvement. Recently, other genome editing techniques such as CRISPR-directed evolution, CRISPR-Cas12a, and base editors have also been used for efficient genome editing in rice. Since rice is an excellent model system for functional studies due to its small genome and close syntenic relationships with other cereal crops, new genome-editing technologies continue to be developed for use in rice. In this review, we focus on genome-editing tools for rice improvement to address current challenges and provide examples of genome editing in rice. We also shed light on expanding the scope of genome editing and systems for delivering homology-directed repair templates. Finally, we discuss safety concerns and methods for obtaining transgene-free crops.

Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C-toT base editing in plants, we first compared seven cytidine deaminases in the BE3like configuration in rice. We found A3A/Y130F-CBE_V01 resulted in the highest C-toT base editing efficiency in both rice and Arabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9-mediated C-toT base editing at Arich PAMs. To showcase its applications, we first applied A3A/Y130F-CBE_V01 for multiplexed editing to generate microRNA-resistant mRNA transcripts as well as premature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F-CBE_V01 for efficient artificial evolution of novel ALS and EPSPS alleles which conferred herbicide resistance in rice. To further improve C-toT base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole-genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome-wide off-target effects in rice. Different levels of cytidine deaminase-dependent and sgRNA-independent off-target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome-wide sgRNA-dependent off-target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1-CBE_V04 and A3A/ Y130F-CBE_V04 as next-generation plant CBEs with high editing efficiency, purity, and specificity.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system was initially discovered as an underlying mechanism for conferring adaptive immunity to bacteria and archaea against viruses. Over the past decade, this has been repurposed as a genome-editing tool. Numerous gene editing-based crop improvement technologies involving CRISPR/Cas platforms individually or in combination with next-generation sequencing methods have been developed that have revolutionized plant genome-editing methodologies. Initially, CRISPR/Cas nucleases replaced the earlier used sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), to address the problem of associated off-targets. The adaptation of this platform led to the development of concepts such as epigenome editing, base editing, and prime editing. Epigenome editing employed epi-effectors to manipulate chromatin structure, while base ed...

One promising application of CRISPR/Cas9 is to create targeted mutations to introduce traits of interest into domesticated organisms. However, a major current limitation for crop and livestock improvement is to identify the precise genes and genetic changes that must be engineered to obtain traits of interest. Here, we discuss the advantages of bio-inspired genome editing, i.e. the engineered introduction of natural mutations that have already been associated with traits of interest in other lineages (breeds, populations or species). To obtain a landscape view of potential targets for genome editing, we used Gephebase (www.gephebase.org), a manually curated database compiling published data about the genes responsible for evolutionary and domesticated changes across eukaryotes, and examined the >1200 mutations that have been identified in the coding regions of more than 700 genes in animals, plants and yeasts. We observe that our genetic knowledge is relatively important for cert...

T he flow of genetic information in living systems is encapsulated by the central dogma of molecular biology: DNA is transcribed into RNA, which is then translated into protein. In reality, of course, the story is much more complex and interesting. But fundamentally, this is us.

The prokaryote-derived Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas mediated gene editing tools have revolutionized our ability to precisely manipulate specific genome sequences in plants and animals. The simplicity, precision, affordability, and robustness of this technology have allowed a myriad of genomes from a diverse group of plant species to be successfully edited. Even though CRISPR/Cas, base editing, and prime editing technologies have been rapidly adopted and implemented in plants, their editing efficiency rate and specificity varies greatly. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9-derived technologies and their implications on enhancing editing efficiency. We highlight the major efforts of engineering Cas9, Cas12a, Cas12b, and Cas12f proteins aiming to improve their efficiencies. We also provide a perspective on the global future of agriculturally based products using DNA-free CRISPR/Cas techniques. The impr...

Modern genome editing (GE) techniques, which include clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and LAGLIDADG homing endonucleases (meganucleases), have so far been used for engineering disease resistance in crops. The use of GE technologies has grown very rapidly in recent years with numerous examples of targeted mutagenesis in crop plants, including gene knockouts, knockdowns, modifications, and the repression and activation of target genes. CRISPR/Cas9 supersedes all other GE techniques including TALENs and ZFNs for editing genes owing to its unprecedented efficiency, relative simplicity and low risk of off-target effects. Broad-spectrum disease resistance has been engineered in crops by GE of either specific host-susceptibility genes (S gene approach), or cleaving DNA of phytopathogens (bacteria, virus or fungi) to inhibit their proliferation. This review focuses on different GE techniques that can potentially be used to boost molecular immunity and resistance against different phytopathogens in crops, ultimately leading to the development of promising disease-resistant crop varieties.

Environmental abiotic stresses challenge food security by depressing crop yields often exceeding 50% of their annual production. Different methods, including conventional as well as genomic-assisted breeding, mutagenesis, and genetic engineering have been utilized to enhance stress resilience in several crop species. Plant breeding has been partly successful in developing crop varieties against abiotic stresses owning to the complex genetics of the traits as well as the narrow genetic base in the germplasm. Irrespective of the fact that genetic engineering can transfer gene(s) from any organism(s), transgenic crops have become controversial mainly due to the potential risk of transgene-outcrossing. Consequently, the cultivation of transgenic crops is banned in certain countries, particularly in European countries. In this scenario, the discovery of the CRISPR tool provides a platform for producing transgene-free genetically edited plants—similar to the mutagenized crops that are not...

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system was initially discovered as an underlying mechanism for conferring adaptive immunity to bacteria and archaea against viruses. Over the past decade, this has been repurposed as a genome-editing tool. Numerous gene editing-based crop improvement technologies involving CRISPR/Cas platforms individually or in combination with next-generation sequencing methods have been developed that have revolutionized plant genome-editing methodologies. Initially, CRISPR/Cas nucleases replaced the earlier used sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), to address the problem of associated off-targets. The adaptation of this platform led to the development of concepts such as epigenome editing, base editing, and prime editing. Epigenome editing employed epi-effectors to manipulate chromatin structure, while base ed...

An ongoing challenge in functional epigenomics is to develop tools for precise manipulation of epigenetic marks. These tools would allow moving from correlation-based to causal-based findings, a necessary step to reach conclusions on mechanistic principles. In this review, we describe and discuss the advantages and limits of tools and technologies developed to impact epigenetic marks, and which could be employed to study their direct effect on nuclear and chromatin structure, on transcription, and their further genuine role in plant cell fate and development. On one hand, epigenome-wide approaches include drug inhibitors for chromatin modifiers or readers, nanobodies against histone marks or lines expressing modified histones or mutant chromatin effectors. On the other hand, locus-specific approaches consist in targeting precise regions on the chromatin, with engineered proteins able to modify epigenetic marks. Early systems use effectors in fusion with protein domains that recogniz...