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Restored Collagen VI Microfilaments Network in the Extracellular Matrix of CRISPR-Edited Ullrich Congenital Muscular Dystrophy Fibroblasts.
Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient's dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient's fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient's skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts.
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CRISPR-associated nucleases: the Dawn of a new age of efficient crop improvement.
The world stands at a new threshold today. As a planet, we face various challenges, and the key one is how to continue to produce enough food, feed, fiber, and fuel to support the burgeoning population. In the past, plant breeding and the ability to genetically engineer crops contributed to increasing food production. However, both approaches rely on random mixing or integration of genes, and the process can be unpredictable and time-consuming. Given the challenge of limited availability of natural resources and changing environmental conditions, the need to rapidly and precisely improve crops has become urgent. The discovery of CRISPR-associated endonucleases offers a precise yet versatile platform for rapid crop improvement. This review summarizes a brief history of the discovery of CRISPR-associated nucleases and their application in genome editing of various plant species. Also provided is an overview of several new endonucleases reported recently, which can be utilized for editing of specific genes in plants through various forms of DNA sequence alteration. Genome editing, with its ever-expanding toolset, increased efficiency, and its potential integration with the emerging synthetic biology approaches hold promise for efficient crop improvement to meet the challenge of supporting the needs of future generations.
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Engineering salt-tolerant Cas12f1 variants for gene-editing applications.
CRISPR has revolutionized the field of genome editing in life sciences by serving as a versatile and state-of-the-art tool. Cas12f1 is a small nuclease of the bacterial immunity CRISPR system with an ideal size for cellular delivery, in contrast to CRISPR-associated (Cas) proteins like Cas9 or Cas12. However, Cas12f1 works best at low salt concentrations. In this study, we find that the plasticity of certain Cas12f1 regions (K196-Y202 and I452-L515) is negatively affected by increased salt concentrations. On this line, key protein domains (REC1, WED, Nuc, lid) that are involved in the DNA-target recognition and the activation of the catalytic RuvC domain are in turn also affected. We suggest that salt concentration should be taken in to consideration for activity assessments of Cas engineered variants, especially if the mutations are on the protospacer adjacent motif interacting domain. The results can be exploited for the engineering of Cas variants and the assessment of their activity at varying salt concentrations. We propose that the K198Q mutation can restore at great degree the compromised plasticity and could potentially lead to salt-tolerant Cas12f1 variants. The methodology can be also employed for the study of biomolecules in terms of their salinity tolerance.Communicated by Ramaswamy H. Sarma.
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"Primed for Repair: Harnessing Hypoxia, Mechanobiology, and Gene Editing to Enhance MSC Potency and Clinical Translation".
Mesenchymal Stem/Stromal Cells (MSCs) hold substantial promise for regenerative medicine; however, their clinical translation has been limited by inconsistent efficacy, poor survival after transplantation, and highly variable functional potency. Preconditioning, defined as the ex vivo manipulation of MSCs before administration, has emerged as a key strategy to address these shortcomings. This review catalogues and critically evaluates the major preconditioning techniques developed between 2000 and 2025, aimed at improving the overall fitness of undifferentiated MSCs or steering their commitment toward specific therapeutic lineages, including osteogenic, chondrogenic, adipogenic, cardiomyogenic, and neurogenic outcomes. We grouped these strategies into two broad categories: (1) non-genomic manipulations, which rely on transient exposure to physical, chemical, or biological cues, such as hypoxia, mechanical forces, small molecules, cytokine treatment, and extracellular matrix engineering; and (2) genomic or gene-modifying manipulations, which involve more stable alterations through microRNA/siRNA modulation, CRISPR/Cas-based gene editing, and viral or non-viral gene transfer. For each modality, we synthesized the relevant molecular mechanisms, evaluated evidence from in vitro, preclinical, and clinical studies, and examined key translational barriers. We also considered important evaluation metrics, including safety, engraftment efficiency, and functional recovery, across disease models. Finally, we integrate perspectives on the evolving clinical trial landscape, regulatory developments from the FDA and EMA, and the intellectual property environment, concluding with a discussion of future research priorities that may accelerate the development of next-generation, potency-enhanced MSC therapeutics.
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Genome Editing to Produce Knockout Mutations of Seed Dormancy Genes in Wheat.
Knockout mutants provide definitive information about the functions of genes related to agronomic traits, including seed dormancy. However, it takes many years to produce knockout mutants using conventional techniques in polyploid plants such as hexaploid wheat. Genome editing with sequence-specific nucleases is a promising approach for obtaining knockout mutations in all targeted homoeologs of wheat simultaneously. Here, we describe a procedure to produce a triple recessive mutant in wheat via genome editing. This protocol covers the evaluation of gRNA and Agrobacterium-mediated transformation to obtain edited wheat seedlings.
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High-Throughput Imaging of CRISPR- and Recombinant Adeno-Associated Virus-Induced DNA Damage Response in Human Hematopoietic Stem and Progenitor Cells.
CRISPR-Cas technology has revolutionized gene editing, but concerns remain due to its propensity for off-target interactions. This, combined with genotoxicity related to both CRISPR-Cas9-induced double-strand breaks and transgene delivery, poses a significant liability for clinical genome-editing applications. Current best practice is to optimize genome-editing parameters in preclinical studies. However, quantitative tools that measure off-target interactions and genotoxicity are costly and time-consuming, limiting the practicality of screening large numbers of potential genome-editing reagents and conditions. Here, we show that flow-based imaging facilitates DNA damage characterization of hundreds of human hematopoietic stem and progenitor cells per minute after treatment with CRISPR-Cas9 and recombinant adeno-associated virus serotype 6. With our web-based platform that leverages deep learning for image analysis, we find that greater DNA damage response is observed for guide RNAs with higher genome-editing activity, differentiating even single on-target guide RNAs with different levels of off-target interactions. This work simplifies the characterization and screening process of genome-editing parameters toward enabling safer and more effective gene-therapy applications.
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CRISPR/CAS9-Mediated Antiviral Activity: A Tool to Combat Viral Infection.
Viruses hijack host cellular receptors and functions for replication, thereby posing a complication in identifying therapeutic targets. The CRISPR/Cas 9 platform has revolutionized gene-editing modalities in a wide range of cells and organisms with high potential in therapeutics. Recently, it has been put to work targeting human pathogenic viruses that interrupt receptors and functions with viral replication. This review encompasses major discoveries in CRISPR/Cas as an antiviral strategy. Additionally, challenges that demand consideration prior to its use in the clinic as part of the antiviral armamentarium are briefly addressed.
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Generation of 3'UTR knockout cell lines by CRISPR/Cas9-mediated genome editing.
In addition to the protein code, messenger RNAs (mRNAs) also contain untranslated regions (UTRs). 3'UTRs span the region between the translational stop codon and the poly(A) tail. Sequence elements located in 3'UTRs are essential for pre-mRNA processing. 3'UTRs also contain elements that can regulate protein abundance, localization, and function. At least half of all human genes use alternative cleavage and polyadenylation (APA) to further diversify the regulatory potential of protein functions. Traditional gene editing approaches are designed to disrupt the production of functional proteins. Here, we describe a method that allows investigators to manipulate 3'UTR sequences of endogenous genes for both single- 3'UTR and multi-3'UTR genes. As 3'UTRs can regulate individual functions of proteins, techniques to manipulate 3'UTRs at endogenous gene loci will help to disentangle multi-functionality of proteins. Furthermore, the ability to directly examine the impact of gene regulatory elements in 3'UTRs will provide further insights into their functional significance.
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CRISPR/Cas Systems towards Next-Generation Biosensing.
Beyond its remarkable genome editing ability, the CRISPR/Cas9 effector has also been utilized in biosensing applications. The recent discovery of the collateral RNA cleavage activity of the Cas13a effector has sparked even greater interest in developing novel biosensing technologies for nucleic acid detection and promised significant advances in CRISPR diagnostics. Now, along with the discovery of Cas12 collateral cleavage activities on single-stranded DNA (ssDNA), several CRISPR/Cas systems have been established for detecting various targets, including bacteria, viruses, cancer mutations, and others. Based on key Cas effectors, we provide a detailed classification of CRISPR/Cas biosensing systems and propose their future utility. As the field continues to mature, CRISPR/Cas systems have the potential to become promising candidates for next-generation diagnostic biosensing platforms.
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Coordinated gene upregulation in maize through CRISPR/Cas-mediated enhancer insertion.
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