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Progress of CRISPR-based programmable RNA manipulation and detection.
Prokaryotic clustered regularly interspaced short palindromic repeats and CRISPR associated (CRISPR-Cas) systems provide adaptive immunity by using RNA-guided endonucleases to recognize and eliminate invading foreign nucleic acids. Type II Cas9, type V Cas12, type VI Cas13, and type III Csm/Cmr complexes have been well characterized and developed as programmable platforms for selectively targeting and manipulating RNA molecules of interest in prokaryotic and eukaryotic cells. These Cas effectors exhibit remarkable diversity of ribonucleoprotein (RNP) composition, target recognition and cleavage mechanisms, and self discrimination mechanisms, which are leveraged for various RNA targeting applications. Here, we summarize the current understanding of mechanistic and functional characteristics of these Cas effectors, give an overview on RNA detection and manipulation toolbox established so far including knockdown, editing, imaging, modification, and mapping RNA-protein interactions, and discuss the future directions for CRISPR-based RNA targeting tools. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
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The role of the CRISPR-Cas system in cancer drug development: Mechanisms of action and therapy.
BACKGROUND: The recent emergence of gene editing using Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated system (Cas) tools and advances in genomics and proteomics has revolutionized drug discovery and personalized medicine. PURPOSE AND SCOPE: The CRISPR-Cas system has enabled gene and cell-based therapies, screening for novel drug targets, a new generation of disease models, elucidation of drug resistance mechanisms, and drug efficacy testing. Here, we summarized recent investigations and strategies involved in cancer-related drug discovery using the CRISPR-Cas system. CONCLUSION: CRISPR-Cas-mediated gene editing has shown great potential in the development of next generation drugs for treatment of Mendelian disorders and various cancer types. In this review, we focused on the impact of the CRISPR-Cas system in drug discovery and its application to biomarker identification and validation, high-end target genes, and breakthrough anticancer cell therapies. We also highlighted the role of CRISPR-Cas in precision disease modeling and functional drug screening.
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Expanding Gene-Editing Potential in Crop Improvement with Pangenomes.
Pangenomes aim to represent the complete repertoire of the genome diversity present within a species or cohort of species, capturing the genomic structural variance between individuals. This genomic information coupled with phenotypic data can be applied to identify genes and alleles involved with abiotic stress tolerance, disease resistance, and other desirable traits. The characterisation of novel structural variants from pangenomes can support genome editing approaches such as Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein Cas (CRISPR-Cas), providing functional information on gene sequences and new target sites in variant-specific genes with increased efficiency. This review discusses the application of pangenomes in genome editing and crop improvement, focusing on the potential of pangenomes to accurately identify target genes for CRISPR-Cas editing of plant genomes while avoiding adverse off-target effects. We consider the limitations of applying CRISPR-Cas editing with pangenome references and potential solutions to overcome these limitations.
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Genome editing tools based improved applications in macrofungi.
Macrofungi commonly referred to as Mushrooms are distributed worldwide and well known for their nutritional, medicinal, and organoleptic properties. Strain improvement in mushrooms is lagging due to paucity of efficient genome modification techniques. Thus, for advanced developments in research and commercial or economical viability and benefit, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) emerged as an efficient genome editing tool. The higher efficiency and precision of the desired genetic modification(s) are the most valuable attributes of this recent technology. The present review comprehensively summarizes various conventional methods utilized for strain improvement in mushrooms including hybridization, protoplast fusion, and di-mon mating. Furthermore, the problems associated with these techniques have been discussed besides providing the potential recluses. The significance of CRISPR/Cas9 strategies employed for improvement in various mushroom genera has been deliberated, as these strategies will paves the way forward for obtaining improved strain and effective cultivation methods for enhancing the yield and quality of the fruit bodies.
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New Advances in Using Virus-like Particles and Related Technologies for Eukaryotic Genome Editing Delivery.
The designer nucleases, including Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas), have been widely used for mechanistic studies, animal model generation, and gene therapy development. Clinical trials using designer nucleases to treat genetic diseases or cancers are showing promising results. Despite rapid progress, potential off-targets and host immune responses are challenges to be addressed for in vivo uses, especially in clinical applications. Short-term expression of the designer nucleases is necessary to reduce both risks. Currently, delivery methods enabling transient expression of designer nucleases are being pursued. Among these, virus-like particles as delivery vehicles for short-term designer nuclease expression have received much attention. This review will summarize recent developments in using virus-like particles (VLPs) for safe delivery of gene editing effectors to complement our last review on the same topic. First, we introduce some background information on how VLPs can be used for safe and efficient CRISPR/Cas9 delivery. Then, we summarize recently developed virus-like particles as genome editing vehicles. Finally, we discuss applications and future directions.
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The application of the CRISPR-Cas system in Pseudomonas aeruginosa infections.
Due to the extensive drug resistance of Pseudomonas aeruginosa (P. aeruginosa), it is still a great clinical challenge. The clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) system has become a promising strategy against this pathogen. This review critically evaluates the multifaceted applications of CRISPR-Cas technology in P. aeruginosa, including its role in antimicrobial resistance, diagnostics, genome editing, and emerging therapeutic and vaccine strategies. In addition to conducting a comprehensive analysis of various studies, we also compared the performance and limitations of various CRISPR platforms, and discussed the main technologies and transformation obstacles in this field. Finally, we look forward to the direction of applying these experimental tools to clinical research in the future.
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Recent advances in CRISPR-Cas system for Saccharomyces cerevisiae engineering.
Yeast Saccharomyces cerevisiae (S. cerevisiae) is a crucial industrial platform for producing a wide range of chemicals, fuels, pharmaceuticals, and nutraceutical ingredients. It is also commonly used as a model organism for fundamental research. In recent years, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) system has become the preferred technology for genetic manipulation in S. cerevisiae owing to its high efficiency, precision, and user-friendliness. This system, along with its extensive toolbox, has significantly accelerated the construction of pathways, enzyme optimization, and metabolic engineering in S. cerevisiae. Furthermore, it has allowed researchers to accelerate phenotypic evolution and gain deeper insights into fundamental biological questions, such as genotype-phenotype relationships. In this review, we summarize the latest advancements in the CRISPR-Cas toolbox for S. cerevisiae and highlight its applications in yeast cell factory construction and optimization, enzyme and phenotypic evolution, genome-scale functional interrogation, gene drives, and the advancement of biotechnologies. Finally, we discuss the challenges and potential for further optimization and applications of the CRISPR-Cas system in S. cerevisiae.
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Ribonucleoproteins Mediated Efficient In Vivo Gene Editing in Skin Stem Cells.
The clustered regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system functions like an adaptive immune system in a variety of microbes and has recently been engineered as a powerful tool for manipulating genomic sequences in a huge variety of cell types. In mammals, CRISPR/Cas9 has the potential to bring curative therapies to patients with genetic diseases, although it remained unknown whether suitable in vivo methods for its use are feasible. It is now appreciated that the efficient delivery of these genome-editing tools into most tissue types, including skin, remains a major challenge. Here, we describe a detailed protocol for performing in vivo gene editing of genomic sequences in mouse skin stem cells using Cas9/sgRNAs ribonucleoproteins in combination with electrotransfer technology. We here present all of the required methods needed for the protocol, including molecular cloning, in vitro sgRNA expression and sgRNA purification, Cas9 protein purification, and in vivo delivery of cas9 ribonucleoproteins. This protocol provides a novel in vivo gene editing strategy using ribonucleoproteins for skin stem cells and can potentially be used as curative treatment for genetic diseases in skin and other somatic tissues.
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Complete Acid Ceramidase ablation prevents cancer-initiating cell formation in melanoma cells.
Acid ceramidase (AC) is a lysosomal cysteine hydrolase that catalyzes the conversion of ceramide into fatty acid and sphingosine. This reaction lowers intracellular ceramide levels and concomitantly generates sphingosine used for sphingosine-1-phosphate (S1P) production. Since increases in ceramide and consequent decreases of S1P reduce proliferation of various cancers, AC might offer a new target for anti-tumor therapy. Here we used CrispR-Cas9-mediated gene editing to delete the gene encoding for AC, ASAH1, in human A375 melanoma cells. ASAH1-null clones show significantly greater accumulation of long-chain saturated ceramides that are substrate for AC. As seen with administration of exogenous ceramide, AC ablation blocks cell cycle progression and accelerates senescence. Importantly, ASAH1-null cells also lose the ability to form cancer-initiating cells and to undergo self-renewal, which is suggestive of a key role for AC in maintaining malignancy and self-renewal of invasive melanoma cells. The results suggest that AC inhibitors might find therapeutic use as adjuvant therapy for advanced melanoma.
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CRISPR/Cas 9-Based Editing in the Production of Bioactive Molecules.
Plants, fungi, and bacteria synthesize a wide range of secondary metabolites that exhibit diverse biological activities. These bioactives, due to their potential benefits in research and therapeutics, have gained immense industrial importance. There is a need to synthesize these bioactives at significantly higher concentrations using cost-effective measures to be economically viable. However, the broader study of industrially important secondary metabolites has been hindered, thus, far due to a shortage of reliable, comparatively easy, and highly effective gene manipulation techniques. With the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas), there is a revolution in the field of genetic engineering. CRISPR/Cas system, due to its simplicity and ease of use. This has widened its application in plant breeding, strain improvement, and engineering the metabolic pathways involved in the biochemical synthesis of industrially valuable bioactive. This review briefly introduces the CRISPR/Cas9 system and summarizes the applications of CRISPR/Cas9-mediated editing tools for the production of plant and fungal-derived bioactives.
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