Biomedical research has witnessed significant strides in manufacturing chimeric antigen receptor T cell (CAR-T) therapies, marking a transformative era in cellular immunotherapy. Nevertheless, existing manufacturing methods for autologous cell therapies still pose several challenges related to cost, immune cell source, safety risks, and scalability. These challenges have motivated recent efforts to optimize process development and manufacturing for cell therapies using automated closed-system bioreactors and models created using artificial intelligence. Simultaneously, non-viral gene transfer methods like mRNA, CRISPR genome editing, and transposons are being applied to engineer T cells and other immune cells like macrophages and natural killer cells. Alternative sources of primary immune cells and stem cells are being developed to generate universal, allogeneic therapies, signaling a shift away from the current autologous paradigm. These multifaceted innovations in manufacturing underscore a collective effort to propel this therapeutic approach toward broader clinical adoption and improved patient outcomes in the evolving landscape of cancer treatment. Here, we review current CAR immune cell manufacturing strategies and highlight recent advancements in cell therapy scale-up, automation, process development, and engineering.

UNASSIGNED: Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal blood disorder, resulting from severe deficiency of plasma ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13) activity. ADAMTS13 is crucial for normal hemostasis through proteolytic cleavage of ultra large von Willebrand factor (VWF). Since the discovery of ADAMTS13 in 2001, several animal models for TTP have been established. In this narrative review, we summarize the creation and characterization of the established animal models for TTP to date.
UNASSIGNED: We performed a literature search through PubMed from 1969 to 2022 using free text: TTP and animal model. We found 67 peer-reviewed articles but only 33 articles were included for review and 34 articles that did not discuss TTP were excluded.
UNASSIGNED: There were genetically modified or antibody-mediated TTP models being established and fully characterized in mouse, rat, baboon, and zebrafish. However, we are still in urgent need of a true autoimmune TTP animal model.
UNASSIGNED: These animal models allowed researchers to further evaluate the contribution of various potential environmental factors and/or genetic modifiers to the pathogenesis, progression, and outcome of TTP; and to help assess the efficacy and safety of novel approaches for prevention and treatment of both hereditary and acquired TTP.

Cancer continues to present a substantial global health challenge, with its incidence and mortality rates persistently reflecting its significant impact. The emergence of precision oncology has provided a breakthrough in targeting oncogenic drivers previously deemed \"undruggable\" by conventional therapeutics and by limiting off-target cytotoxicity. Two groundbreaking technologies that have revolutionized the field of precision oncology are primarily CRISPR-Cas9 gene editing and more recently PROTAC (PROteolysis TArgeting Chimeras) targeted protein degradation technology. CRISPR-Cas9, in particular, has gained widespread recognition and acclaim due to its remarkable ability to modify DNA sequences precisely. Rather than editing the genetic code, PROTACs harness the ubiquitin proteasome degradation machinery to degrade proteins of interest selectively. Even though CRISPR-Cas9 and PROTAC technologies operate on different principles, they share a common goal of advancing precision oncology whereby both approaches have demonstrated remarkable potential in preclinical and promising data in clinical trials. CRISPR-Cas9 has demonstrated its clinical potential in this field due to its ability to modify genes directly and indirectly in a precise, efficient, reversible, adaptable, and tissue-specific manner, and its potential as a diagnostic tool. On the other hand, the ability to administer in low doses orally, broad targeting, tissue specificity, and controllability have reinforced the clinical potential of PROTAC. Thus, in the field of precision oncology, gene editing using CRISPR technology has revolutionized targeted interventions, while the emergence of PROTACs has further expanded the therapeutic landscape by enabling selective protein degradation. Rather than viewing them as mutually exclusive or competing methods in the field of precision oncology, their use is context-dependent (i.e., based on the molecular mechanisms of the disease) and they potentially could be used synergistically complementing the strengths of CRISPR and vice versa. Herein, we review the current status of CRISPR and PROTAC designs and their implications in the field of precision oncology in terms of clinical potential, clinical trial data, limitations, and compare their implications in precision clinical oncology.

Cyanobacteria are important primary producers, contributing to 25% of the global carbon fixation through photosynthesis. They serve as model organisms to study the photosynthesis, and are important cell factories for synthetic biology. To enable efficient genetic dissection and metabolic engineering in cyanobacteria, effective and accurate genetic manipulation tools are required. However, genetic manipulation in cyanobacteria by the conventional homologous recombination-based method and the recently developed CRISPR-Cas gene editing system require complicated cloning steps, especially during multi-site editing and single base mutation. This restricts the extensive research on cyanobacteria and reduces its application potential. In this study, a highly efficient and convenient cytosine base editing system was developed which allows rapid and precise C → T point mutation and gene inactivation in the genomes of Synechocystis and Anabaena. This base editing system also enables efficient multiplex editing and can be easily cured after editing by sucrose counter-selection. This work will expand the knowledge base regarding the engineering of cyanobacteria. The findings of this study will encourage the biotechnological applications of cyanobacteria.

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The efficient natural transformation of Neisseria meningitidis allows the rapid construction of bacterial mutants in which the genes of interest are interrupted or replaced by antibiotic-resistance cassettes. However, this proved to be a double-edged sword, i.e., although facilitating the genetic characterization of this important human pathogen, it has limited the development of strategies for constructing markerless mutants without antibiotic-resistance markers. In addition, efficient tools for complementation or labeling are also lacking in N. meningitidis. In this study, we significantly expand the meningococcal genetic toolbox by developing new and efficient tools for the construction of markerless mutants (using a dual counterselection strategy), genetic complementation (using integrative vectors), and cell labeling (using a self-labeling protein tag). This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.IMPORTANCENeisseria meningitidis and Neisseria gonorrhoeae are two important human pathogens. Research focusing on these bacteria requires genetic engineering, which is facilitated by their natural ability to undergo transformation. However, the ease of mutant engineering has led the Neisseria community to neglect the development of more sophisticated tools for gene editing, particularly for N. meningitidis. In this study, we have significantly expanded the meningococcal genetic toolbox by developing novel and efficient tools for markerless mutant construction, genetic complementation, and cell tagging. This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.

One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research.

Livestock production significantly contributes to greenhouse gas (GHG) emissions particularly methane (CH4) emissions thereby influencing climate change. To address this issue further, it is crucial to establish strategies that simultaneously increase ruminant productivity while minimizing GHG emissions, particularly from cattle, sheep, and goats. Recent advancements have revealed the potential for modulating the rumen microbial ecosystem through genetic selection to reduce methane (CH4) production, and by microbial genome editing including CRISPR/Cas9, TALENs (Transcription Activator-Like Effector Nucleases), ZFNs (Zinc Finger Nucleases), RNA interference (RNAi), Pime editing, Base editing and double-stranded break-free (DSB-free). These technologies enable precise genetic modifications, offering opportunities to enhance traits that reduce environmental impact and optimize metabolic pathways. Additionally, various nutrition-related measures have shown promise in mitigating methane emissions to varying extents. This review aims to present a future-oriented viewpoint on reducing methane emissions from ruminants by leveraging CRISPR/Cas9 technology to engineer the microbial consortia within the rumen. The ultimate objective is to develop sustainable livestock production methods that effectively decrease methane emissions, while maintaining animal health and productivity.

In recent years, targeted genome editing has emerged as an indispensable tool for creating animal models, facilitating a comprehensive exploration of the molecular mechanisms governing a myriad of biological processes. Within this scientific landscape, the investigation of meiosis in mice has attracted considerable attention across numerous research laboratories. The precision and versatility of the CRISPR/Cas9 genome editing system have revolutionized our ability to generate mice with tailored genetic alterations, including point mutations and null mutations. These genetic modifications have provided invaluable insights into the intricate functionality of various meiotic genes and their associated variants. In this context, we present a detailed state of the art protocol for the creation of novel mouse models, each bearing specific genetic modifications within key meiotic genes, through the application of CRISPR/Cas9 technology. Furthermore, we showcase two distinct genetic modifications, accomplished within our laboratory, that can serve as valuable reference points for researchers seeking to elucidate the molecular intricacies of meiosis in mammals.

Base editing represents a cutting-edge genome editing technique that utilizes the CRISPR system to guide base deaminases with high precision to specific genomic sites, facilitating the targeted alteration of individual nucleotides. Unlike traditional gene editing approaches, base editing does not require DNA double-strand breaks or donor templates. It functions independently of the cellular DNA repair machinery, offering significant advantages in terms of both efficiency and accuracy. In this review, we summarize the core design principles of various DNA base editors, their distinctive editing characteristics, and tactics to refine their efficacy. We also summarize their applications in crop genetic improvement and explore their potential contributions to forest genetic engineering.