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[Genome Editing with CRISPR/Cas9: First Steps Towards a new Era in Medicine?].
The emergence of genome editing technologies can be regarded as one of the most groundbreaking revolutions in the history of science. Modern genome editing allows the introduction of precise mutations into the genome of virtually all cells and organisms without leaving any additional trace. Undoubtedly, genome editing with CRISPR/Cas9, often casually referred to as "genetic scissors", will revolutionize medical research and development. However, at the same time it creates a great need for ethical considerations as it might hold risks for both people and the environment that cannot yet be fully assessed. While genome editing is already well established in laboratory research, clinical applications based on genome editing are close. For the first time, targeted corrections of genetic defects in somatic cells, stem cells as well as in the germ line appear technically feasible. This generates possible future scenarios that urgently require broad ethical and social discussions.
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A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines.
Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.
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Metal coordination micelles for anti-cancer treatment by gene-editing and phototherapy.
CRISPR-Cas9 is a central focus of the emerging field of gene editing and photodynamic therapy (PDT) is a clinical-stage ablation modality combining photosensitizers with light irradiation. But metal coordination biomaterials for the applications of both have rarely been investigated. Herein, Chlorin-e6 (Ce6) Manganese (Mn) coordination micelles loaded with Cas9, termed Ce6-Mn-Cas9, were developed for augmented combination anti-cancer treatment. Manganese played multiple roles to facilitate Cas9 and single guide RNA (sgRNA) ribonucleoprotein (RNP) delivery, Fenton-like effect, and enhanced endonuclease activity of RNP. Histidine (His)-tagged RNP could be coordinated to Ce6 encapsulated in Pluronic F127 (F127) micelles by simple admixture. Triggered by ATP and endolysosomal acidic pH, Ce6-Mn-Cas9 released Cas9 without altering protein structure or function. Dual guide RNAs were designed to target the antioxidant regulator MTH1 and the DNA repair protein APE1, resulting in increased oxygen and enhanced PDT effect. In a murine tumor model, Ce6-Mn-Cas9 inhibited tumor growth with the combination therapy of PDT and gene editing. Taken together, Ce6-Mn-Cas9 represents a new biomaterial with a high degree of versatility to enable photo- and gene-therapy approaches.
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CRISPR-Cas and CRISPR-based screening system for precise gene editing and targeted cancer therapy.
Target cancer therapy has been developed for clinical cancer treatment based on the discovery of CRISPR (clustered regularly interspaced short palindromic repeat) -Cas system. This forefront and cutting-edge scientific technique improves the cancer research into molecular level and is currently widely utilized in genetic investigation and clinical precision cancer therapy. In this review, we summarized the genetic modification by CRISPR/Cas and CRISPR screening system, discussed key components for successful CRISPR screening, including Cas enzymes, guide RNA (gRNA) libraries, target cells or organs. Furthermore, we focused on the application for CAR-T cell therapy, drug target, drug screening, or drug selection in both ex vivo and in vivo with CRISPR screening system. In addition, we elucidated the advantages and potential obstacles of CRISPR system in precision clinical medicine and described the prospects for future genetic therapy.In summary, we provide a comprehensive and practical perspective on the development of CRISPR/Cas and CRISPR screening system for the treatment of cancer defects, aiming to further improve the precision and accuracy for clinical treatment and individualized gene therapy.
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Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo.
Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.
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Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6.
Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs). In addition, the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR. We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks, but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics.
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CRISPR/Cas9-directed epigenetic editing in colorectal cancer.
Colorectal cancer (CRC) remains a leading cause of cancer-related illness and death worldwide, arising from a complex interplay of genetic predisposition, environmental influences, and epigenetic dysregulation. Among these factors, epigenetic modifications-reversible and heritable changes in gene expression-serve as crucial regulators of CRC progression. Understanding these modifications is essential for identifying potential biomarkers for early diagnosis and developing targeted therapeutic strategies. Epigenetic drugs (epidrugs) such as DNA methyltransferase inhibitors (e.g., decitabine) and bromodomain inhibitors (e.g., JQ1) have shown promise in modulating aberrant epigenetic changes in CRC. However, challenges such as drug specificity, delivery, and safety concerns limit their clinical application. Advances in CRISPR-Cas9-based epigenetic editing offer a more precise approach to modifying specific epigenetic markers, presenting a potential breakthrough in CRC treatment. Despite its promise, CRISPR-based epigenome editing may result in unintended genetic modifications, necessitating stringent regulations and safety assessments. Beyond pharmacological interventions, lifestyle factors-including diet and gut microbiome composition-play a significant role in shaping the epigenetic landscape of CRC. Nutritional and microbiome-based interventions have shown potential in preventing CRC development by maintaining intestinal homeostasis and reducing tumor-promoting epigenetic changes. This review provides a comprehensive overview of epigenetic alterations in CRC, exploring their implications for diagnosis, prevention, and treatment. By integrating multi-omics approaches, single-cell technologies, and model organism studies, future research can enhance the specificity and efficacy of epigenetic-based therapies. Shortly, a combination of advanced gene-editing technologies, targeted epidrugs, and lifestyle interventions may pave the way for more effective and personalized CRC treatment strategies.
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Rising from the dead: the power of genome editing.
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Genome editing in plants: New advances and applications in plant biology and agriculture.
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Gene Editing: Powerful New Tools for Nephrology Research and Therapy.
Biologic research is experiencing a transformation brought about by the ability of programmable nucleases to manipulate the genome. In the recently developed CRISPR/Cas system, short RNA sequences guide the endonuclease Cas9 to any location in the genome, causing a DNA double-strand break (DSB). Repair of DSBs allows the introduction of targeted genetic manipulations with high precision. Cas9-mediated gene editing is simple, scalable, and rapid, and it can be applied to virtually any organism. Here, we summarize the development of modern gene editing techniques and the biology of DSB repair on which these techniques are based. We discuss technical points in applying this technology and review its use in model organisms. Finally, we describe prospects for the use of gene editing to treat human genetic diseases. This technology offers tremendous promise for equipping the nephrology research community to better model and ultimately, treat kidney diseases.
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