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[Advances in gene therapy for β-thalassemia and hemophilia based on the CRISPR/Cas9 technology].
Thalassemia and hemophilia are common inherited blood disorders caused by genetic abnormalities. These diseases are difficult to cure and can be inherited to the next generation, causing severe family and social burden. The emergence of gene therapy provides a new treatment for genetic diseases. However, since its first clinical trial in 1990, the development of gene therapy has not been as optimistic in the past three decades as one could hope. The development of gene-editing technology, particularly the third generation gene-editing technology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), has given hope in such therapeutic approach for having advantages in high editing efficiency, simple operation, and low cost. Gene editing-mediated gene therapy has thus received increasing attention from the biomedical community. It has shown promises for the treatment of inherited blood disorders, such as thalassemia and hemophilia. This paper reviews the fundamental research progress of gene therapy for β-thalassemia and hemophilia based on CRISPR/Cas9 technology in the past six years. It also summarizes the CRISPR/Cas9-based clinical trials of gene therapy. The problems and possible solutions to this technology for gene therapy are also discussed, thereby providing a reference for the research on gene therapy of inherited blood disorders based on CRISPR/Cas9 technology.
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Comparison of the Off-Target Effects Among One-Base to Three-Base Mismatched Targets of gRNA Using a Blue to White Assay.
The present study was designed to test a new strategy for comparing the off-target effects of CRISPR/cas9 employing a blue/white colony based assay. Eight types of AmpR plasmids with matched, one base mismatched, two bases mismatched and three bases mismatched targets were constructed. The wild typed alpha peptide of the beta-glactosidase gene and some with mutations, chosen as the targets, were successfully subcloned into the plasmids in an inframe pattern. The relevant gRNA and cas9 were subcloned into the ChloR plasmid. Transformation of the plasmid harboring the target gene yielded blue colonies, while white colonies formed in the plates following the co-transformation of target gene plasmid and the CRISPR/cas9 plasmid. Colonies in different levels of blue color were formed from the plasmids having completely matched target and mismatched targets. The appearance of different blue levels following the co-transformation of the different target plasmid and CRISPR/cas9 plasmid qualitatively indicated the effectiveness of the gRNA, while the ratio of the AMP/CL colonies can be used for relatively quantitative evaluation. The target and off-target effects among different targets of gRNA were differentially identifiable and these effects in turn demonstrated the effectiveness of the blue to white assay. Either the qualitative or the quantitative analysis of the blue to white depends on the final interactions among the target, the cas9 and the gRNA. Clearly, when co-transformation is replaced by single transformation using a specialized nanoparticle containing these three components, the target and off-target effect will be more accurately evaluated. Thus, this study provided a model for application of nanoparticle in gene editing studies.
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CRISPR-Cas9 system-driven site-specific selection pressure on Herpes simplex virus genomes.
The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system has been widely used for viral genome editing, transcription regulation and chromosomal localization in eukaryotic cells. In this study, a guide RNA (gRNA) that specifically recognizes HSV-1 viral genomes was used in the CRISPR-Cas9 system to inhibit viral replication. This inhibition could be achieved with both wild type Cas9 protein and Cas9 nickase (D10A). By targeting viral genomes containing sequences recognized by the gRNA, the CRISPR-Cas9 system distinguished between different viral genome sequences and provided single nucleotide-specific selection pressure to significantly change the proportions of viruses in a mixed viral pool. This finding indicates the utility of this tool for virus selection without the need for antibiotics or reporter genes, which could potentially save time compared to other methods used for screening and purifying mutant viruses.
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Exploring retinal degenerative diseases through CRISPR-based screening.
The CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein9) system has emerged as a powerful genetic tool, gaining global recognition as a versatile and efficient gene-editing technique. Its transformation into a high-throughput research platform, CRISPR Screening, has demonstrated wide applicability across various fields such as cancer biology, virology, and drug target discovery, resulting in significant advances. However, its potential in studying retinal degenerative diseases remains largely unexplored, despite the urgent need for effective treatments arising from an incomplete understanding of disease mechanisms. This review aims to present a comprehensive overview of the evolution and current state of CRISPR tools and CRISPR screening methodologies. Noteworthy pioneering studies utilizing these technologies are discussed, alongside experimental design guidelines, including positive and negative selection strategies and delivery methods for sgRNAs (single guide RNAs) and Cas proteins. Furthermore, we explore existing in vitro models appropriate for CRISPR screening in retinal research and identify relevant research questions that could be addressed through this approach. It is anticipated that this review will stimulate innovation in retinal research, facilitating a deeper comprehension of retinal pathophysiology and paving the way for groundbreaking therapeutic interventions and enhanced patient outcomes in the management of retinal degenerative disorders.
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CRISPR/Cas9-mediated single and biallelic knockout of poplar STERILE APETALA (PopSAP) leads to complete reproductive sterility.
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Gene editing for skin diseases: designer nucleases as tools for gene therapy of skin fragility disorders.
What is the topic of this review? This review concerns current gene editing strategies for blistering skin diseases with respect to individual genetic constellations and distinct conditions. What advances does it highlight? Specificity and safety dominate the discussion of gene editing applications for gene therapy, where a number of tools are implemented. Recent developments in this rapidly progressing field pose further questions regarding which tool is best suited for each particular use. The current treatment of inherited blistering skin diseases, such as epidermolysis bullosa (EB), is largely restricted to wound care and pain management. More effective therapeutic strategies are urgently required, and targeting the genetic basis of these severe diseases is now within reach. Here, we describe current gene editing tools and their potential to correct gene function in monogenetic blistering skin diseases. We present the features of the most frequently used gene editing techniques, transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), determining their preferential application for specific genetic conditions, including the type of mutational inheritance, the targeting site within the gene or the possibility to target the mutation specifically. Both tools have traits beneficial in specific situations. Promising developments in the field engender gene editing as a potentially powerful therapeutic option for future clinical applications.
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas genome editing transforming crop stress tolerance for global food security.
Climate change increasingly threatens global crop productivity by intensifying drought, salinity, temperature extremes, and biotic stresses. Developing climate-resilient cultivars has therefore become a central objective in modern crop breeding programs. Conventional breeding approaches are often limited by complex trait inheritance and long selection cycles, particularly for polygenic stress-adaptive traits. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) genome editing genome editing provides a precise and efficient platform for targeted manipulation of genes controlling stress tolerance, yield stability, and adaptive performance. This review synthesizes recent advances in CRISPR mediated improvement of resilience to major abiotic stresses (drought, salinity, heat, and cold) and biotic stresses (fungi, bacteria, viruses, and insects) across important cereal, legume, and horticultural crops. Emphasis is placed on the editing of transcription factors, signaling regulators, susceptibility genes, and redox-associated pathways that enhance physiological and molecular stress adaptation. Furthermore, the integration of CRISPR with genomics, transcriptomics, proteomics, metabolomics, genome-wide association studies, high-throughput phenotyping, and artificial intelligence-driven prediction tools is accelerating precision breeding strategies. Despite remaining challenges related to off-target effects, delivery systems, and regulatory frameworks, genome editing represents a transformative approach for advancing climate-resilient crop development and sustainable agricultural production.
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CRISPR Editing Technology in Biological and Biomedical Investigation.
The CRISPR or clustered regularly interspaced short palindromic repeats system is currently the most advanced approach to genome editing and is notable for providing an unprecedented degree of specificity, effectiveness, and versatility in genetic manipulation. CRISPR evolved as a prokaryotic immune system to provide an acquired immunity and resistance to foreign genetic elements such as bacteriophages. It has recently been developed into a tool for the specific targeting of nucleotide sequences within complex eukaryotic genomes for the purpose of genetic manipulation. The power of CRISPR lies in its simplicity and ease of use, its flexibility to be targeted to any given nucleotide sequence by the choice of an easily synthesized guide RNA, and its ready ability to continue to undergo technical improvements. Applications for CRISPR are numerous including creation of novel transgenic cell animals for research, high-throughput screening of gene function, potential clinical gene therapy, and nongene-editing approaches such as modulating gene activity and fluorescent tagging. In this prospect article, we will describe the salient features of the CRISPR system with an emphasis on important drawbacks and considerations with respect to eliminating off-target events and obtaining efficient CRISPR delivery. We will discuss recent technical developments to the system and we will illustrate some of the most recent applications with an emphasis on approaches to eliminate human viruses including HIV-1, JCV and HSV-1 and prospects for the future. J. Cell. Biochem. 118: 3586-3594, 2017. © 2017 Wiley Periodicals, Inc.
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CRISPR-Powered Liquid Biopsies in Cancer Diagnostics.
Liquid biopsies promise major advantages for cancer screening and diagnosis. By detecting biomarkers in peripheral blood samples, liquid biopsies reduce the need for invasive techniques and provide important genetic information integral to the emerging molecular classification of cancers. Unfortunately, the concentrations of most biomarkers, particularly circulating tumour nucleic acids, are vanishingly small-beyond the sensitivity and specificity of most assays. Clustered Regularly Interspaced Short Palindromic Repeats diagnostics (herein labelled 'CRISPR-Dx') use gene editing tools to detect, rather than modify, nucleic acids with extremely high specificity. These tools are commonly combined with isothermal nucleic acid amplification to also achieve sensitivities comparable to high-performance laboratory-based techniques, such as digital PCR. CRISPR assays, however, are inherently well suited to adaptation for point-of-care (POC) use, and unlike antigen-based POC assays, are significantly easier and faster to develop. In this review, we summarise current CRISPR-Dx platforms and their analytical potential for cancer biomarker discovery, with an emphasis on enhancing early diagnosis, disease monitoring, point-of-care testing, and supporting cancer therapy.
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CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.
Several groups have used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for DNA virus editing. In most cases, one single-guide RNA (sgRNA) is used, which produces inconsistencies in gene editing. In this study, we used a swine herpesvirus, pseudorabies virus, as a model to systematically explore the application of CRISPR/Cas9 in DNA virus editing. In our current report, we demonstrated that cotransfection of 2 sgRNAs and a viral genome resulted in significantly better knockout efficiency than the transfection-infection-based approach. This method could result in 100% knockout of ≤3500 bp of viral nonessential large fragments. Furthermore, knockin efficiency was significantly improved by using 2 sgRNAs and was also correlated with the number of background viruses. We also demonstrated that the background viruses were all 2-sgRNA-mediated knockout mutants. Finally, this study demonstrated that the efficacy of gene knockin is determined by the replicative kinetics of background viruses. We propose that CRISPR/Cas9 coupled with 2 sgRNAs creates a powerful tool for DNA virus editing and offers great potential for future applications.-Tang, Y.-D., Guo, J.-C., Wang, T.-Y., Zhao, K., Liu, J.-T., Gao, J.-C., Tian, Z.-J., An, T.-Q., Cai, X.-H. CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.
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