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Tissue specificity of DNA repair: the CRISPR compass.
CRISPR-Cas9-mediated genome editing holds great promise for the correction of pathogenic variants in humans. However, its therapeutic implementation is hampered due to unwanted editing outcomes. A better understanding of cell type- and tissue-specific DNA repair processes will ultimately enable precise control of editing outcomes for safer and effective therapies.
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Optogenetic gene editing in regional skin.
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Self-limiting population genetic control with sex-linked genome editors.
In male heterogametic species the Y chromosome is transmitted solely from fathers to sons, and is selected for based only on its impacts on male fitness. This fact can be exploited to develop efficient pest control strategies that use Y-linked editors to disrupt the fitness of female descendants. With simple population genetic and dynamic models we show that Y-linked editors can be substantially more efficient than other self-limiting strategies and, while not as efficient as gene drive approaches, are expected to have less impact on non-target populations with which there is some gene flow. Efficiency can be further augmented by simultaneously releasing an autosomal X-shredder construct, in either the same or different males. Y-linked editors may be an attractive option to consider when efficient control of a species is desired in some locales but not others.
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Design of experiments as a decision tool for cell therapy manufacturing.
BACKGROUND AIMS: Cell therapies are costlier to manufacture than small molecules and protein therapeutics because they require multiple manipulations and are often produced in an autologous manner. Strategies to lower the cost of goods to produce a cell therapy could make a significant impact on its total cost. METHODS: Borrowing from the field of bioprocess development, the authors took a design of experiments (DoE)-based approach to understanding the manufacture of a cell therapy product in pre-clinical development, analyzing main cost factors in the production process. The cells used for these studies were autologous CD4+ T lymphocytes gene-edited using CRISPR/Cas9 and recombinant adeno-associated virus (AAV) to restore normal FOXP3 gene expression as a prospective investigational product for patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. RESULTS: Using gene editing efficiency as the response variable, an initial screen was conducted for other variables that could influence the editing frequency. The multiplicity of infection (MOI) of AAV and amount of single guide RNA (sgRNA) were the significant factors used for the optimization step to generate a response contour plot. Cost analysis was done for multiple points in the design space to find cost drivers that could be reduced. For the range of values tested (50 000-750 000 vg/cell AAV and 0.8-4 μg sgRNA), editing with the highest MOI and sgRNA yielded the best gene editing frequency. However, cost analysis showed the optimal solution was gene editing at 193 000 vg/cell AAV and 1.78 μg sgRNA. CONCLUSIONS: The authors used DoE to define key factors affecting the gene editing process for a potential investigational therapeutic, providing a novel and faster data-based approach to understanding factors driving complex biological processes. This approach could be applied in process development and aid in achieving more robust strategies for the manufacture of cellular therapeutics.
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Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9.
CRISPR/Cas9 genome editing technology has been implemented in almost all living organisms. Its editing precision appears to be very high and therefore could represent a big change from conventional genetic engineering approaches. However, guide RNA binding to nucleotides similar to the target site could result in undesired off-target mutations. Despite this, evaluating whether mutations occur is rarely performed in genome editing studies. In this study, we generated CRISPR/Cas9-derived filamentous fungal strains and analyzed them for the occurrence of mutations, and to which extent genome stability affects their occurrence. As a test case, we deleted the (hemi-)cellulolytic regulator-encoding gene xlnR in two Aspergillus niger strains: a wild type (WT) and a non-homologous end-joining (NHEJ)-deficient strain ΔkusA. Initial phenotypic analysis suggested a much higher prevalence of mutations in the WT compared to NHEJ-deficient strains, which was confirmed and quantified by whole-genome sequencing analysis. Our results clearly demonstrate that CRISPR/Cas9 applied to an NHEJ-deficient strain is an efficient strategy to avoid unwanted mutations. IMPORTANCE Filamentous fungi are commonly used biofactories for the production of industrially relevant proteins and metabolites. Often, fungal biofactories undergo genetic development (genetic engineering, genome editing, etc.) aimed at improving production yields. In this context, CRISPR/Cas9 has gained much attention as a genome editing strategy due to its simplicity, versatility, and precision. However, despite the high level of accuracy reported for CRISPR/Cas9, in some cases unintentional cleavages in non-targeted loci-known as off-target mutations-could arise. While biosafety should be a central feature of emerging biotechnologies to minimize unintended consequences, few studies quantitatively evaluate the risk of off-target mutations. This study demonstrates that the use of non-homologous end-joining-deficient fungal strains drastically reduces the number of unintended genomic mutations, ensuring that CRISPR/Cas9 can be safely applied for strain development.
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Efficient CRISPR-Cas genome editing in brown algae.
Brown algae represent the third most complex lineage to have independently evolved multicellularity, distinct from plants and animals. Yet, functional studies of their development and evolution have been limited by the absence of efficient genome editing tools. Here, we present a robust, high-efficiency, and transgene-free CRISPR-based genome editing platform applicable across four ecologically and biotechnologically important brown algal species. Using Ectocarpus as a model, we optimized a polyethylene glycol (PEG)-mediated ribonucleoprotein (RNP) delivery system that achieves reproducible editing across multiple loci without cloning or specialized equipment. As proof of concept, we recreated the hallmark imm mutant phenotype by precisely editing the IMMEDIATE UPRIGHT (IMM) locus. APT/2-fluoroadenine (2-FA) selection further enhanced specificity with minimal false positives. The method was easily transferable to other species, including kelps. This platform now enables functional genomics in brown algae, providing powerful tools for investigating development, life cycle regulation, and the independent evolution of complex multicellularity.
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Bioethics in assisted reproduction and embryology.
The use of assisted reproductive therapies, advances in embryo research, and developments in scientific fields such as gene editing and in vitro gametogenesis have attracted the attention of bioethicists for years. On one side, the 14-day rule has faced criticism from embryo researchers advocating for its extension. On the other side, the increasing number of cryopreserved embryos worldwide has raised practical and ethical concerns about their fate. While advancements in scientific research, especially gene editing and in-vitro gametogenesis (IVG), are not yet fully applicable, their potential future use appears to pose significant bioethical questions. In this review, we examine the evolving bioethics of embryo research, focusing on the 14-day rule, the challenges surrounding surplus cryopreserved embryos, and the future dilemmas posed by CRISPR-based gene editing, IVG, and preimplantation genetic testing for polygenic risk (PGT-P). We also highlight the critical role of multidisciplinary, patient-centered counseling in ART practice, to foster informed consent, realistic expectations, and psychosocial well-being. Finally, we underscore the need for anticipatory ethical frameworks and open-society engagement that integrate public deliberation with scientific progress to ensure that reproductive innovation proceeds responsibly, preserving both human dignity and social justice.
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CRISPR-Cas9 editing of synaptic genes in human embryonic stem cells for functional analysis in induced human neurons.
Generating stable human embryonic stem cells (hESCs) with targeted genetic mutations allows for the interrogation of protein function in numerous cellular contexts while maintaining a relatively high degree of isogenicity. We describe a step-by-step protocol for generating knockout hESC lines with mutations in genes involved in synaptic transmission using CRISPR-Cas9. We describe steps for gRNA design, cloning, stem cell transfection, and clone isolation. We then detail procedures for gene knockout validation and differentiation of stem cells into functional induced neurons.
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Road to the future of systems biotechnology: CRISPR-Cas-mediated metabolic engineering for recombinant protein production.
The rising potential for CRISPR-Cas-mediated genome editing has revolutionized our strategies in basic and practical bioengineering research. It provides a predictable and precise method for genome modification in a robust and reproducible fashion. Emergence of systems biotechnology and synthetic biology approaches coupled with CRISPR-Cas technology could change the future of cell factories to possess some new features which have not been found naturally. We have discussed the possibility and versatile potentials of CRISPR-Cas technology for metabolic engineering of a recombinant host for heterologous protein production. We describe the mechanisms involved in this metabolic engineering approach and present the diverse features of its application in biotechnology and protein production.
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Enhanced precision and efficiency.
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