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Recent developments in CRISPR/Cas9 genome editing research for edible fungiculture.
Fungiculture refers to the deliberate cultivation or agricultural practice involving the growth and management of fungi. The practice encompasses the intentional culture of diverse species of macrofungi, including mushrooms and truffles, within controlled habitats or under specified conditions, in order to fulfill human requirements especially for food purpose. As the global market for edible mushrooms grows quickly, it is becoming increasingly necessary to grow novel and improved strains of edible fungi. Growing and breeding edible fungi using traditional methods is both time-consuming and difficult. So, there is a need for evolving advanced techniques at a molecular level which can help breeding of edible fungi with much better efficiency. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system is one of the most effective techniques for accurately cutting and modifying the genomes of edible fungi. In this review, we discuss how genome editing using CRISPR/Cas9 has been utilized in many edible fungal species such as Pleurotus ostreatus, Agaricus bisporus, Cordyceps militaris, Ganoderma lucidum, Flammulina filiformis, Lentinula edodes, and others for their target specific breeding. We also discuss the working mechanism of the above-mentioned system in these mushroom species, and also the advantages and limitations of using this system in mushrooms.
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Genetic Manipulation Tools in Leishmania: From CRISPR/Cas9 to Vaccine Strategies for Disease Control.
PURPOSE: Gene editing technologies have emerged as a crucial approach totackling parasitic infections. Recent research underscores the potential of protozoan parasites, including Leishmania, to utilize gene editing strategies, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins)(CRISPR/Cas9) to edit parasite genomes, modify virulence factors, and alter host immune responses. This review aims to explore the potential of CRISPR/Cas9 in the study of Leishmania and Leishmaniasis, focusing on its applications in understanding mechanisms and developing novel editing strategies. METHODS: The immunological response of the mammalian host and the nature of the Leishmania parasites play essential roles in the formation and progression of parasitic diseases. Among alternative treatment strategies, CRISPR/Cas9 has attracted attention as a promising tool for introducing protective or therapeutic mutations in Leishmaniasis. This review will provide recent insights into the role of gene editing, especially CRISPR/Cas9, in host-pathogen interactions, intercellular communication, immunomodulation, and pathogenesis of Leishmania. RESULTS: Current findings reveal that CRISPR/Cas9 enables efficient modification of Leishmania genomes, providing valuable insights into parasite biology, host-pathogen interactions, intercellular communication, immunomodulation, and disease pathogenesis. Additionally, immunotherapeutic models are being investigated to explore the potential applications of CRISPR/Cas9 in theranostics. CONCLUSION: CRISPR/Cas9 has attracted attention as a promising tool for introducing protective or therapeutic mutations in Leishmaniasis. Integrating CRISPR/Cas9 with Leishmania-related research opens new avenues for disease control and understanding, while biologically inspired immunomodulatory strategies highlight its emerging role in next-generation parasite-targeted immunotherapy.
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CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells.
Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.
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AZD7648 (DNA-PKcs inhibitor): a two-edged sword for editing genomes.
Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) has been the most practical technique in genome editing for the last decade. Its molecular mechanism includes steps that occur in a sequence, starting from a break in a double strand to repair. After a double-strand break in the DNA strand, the repairing of DNA done via Homology-Directed Repair (HDR) is considered important in different organisms as it is ideal for precise genome editing and the reduction of unintended mutations. Still, it is mostly dominated by the Non-Homologous End Joining (NHEJ) pathway. A recent study by Cullot et al. published in Nature Biotechnology showed interesting features of AZD7648 (a DNA-PKcs inhibitor) that increase the probability of HDR event while DNA repairing (Cullot et al. 2024).
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Evaluation of the immunogenicity of auxotrophic Lactobacillus with CRISPR-Cas9D10A system-mediated chromosomal editing to express porcine rotavirus capsid protein VP4.
Porcine rotavirus (PoRV) is an important pathogen, leading to the occurrence of viral diarrhoea . As the infection displays obvious enterotropism, intestinal mucosal immunity is the significant line of defence against pathogen invasion. Moreover, as lactic acid bacteria (LAB) show acid resistance, bile salt resistance and immune regulation, it is of great significance to develop an oral vaccine. Most traditional plasmid delivery vectors use antibiotic genes as selective markers, easily leading to antibiotic accumulation. Therefore, to select a food-grade marker in genetically engineering food-grade microorganisms is vital. Based on the CRISPR-Cas9D10A system, we constructed a stable auxotrophic Lactobacillus paracasei HLJ-27 (Lactobacillus △Alr HLJ-27) strain. In addition, as many plasmids replicate in the host bacteria, resulting in internal gene deletions. In this study,we used a temperature-sensitive gene editing plasmidto insert the VP4 gene into the genome, yielding the insertion mutant strains VP4/△Alr HLJ-27, VP4/△Alr W56, and VP4/W56. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses. These oral mucosal vaccines have the potential to act as an alternative to the application of antibiotics in the future and induce efficient immune responses against PEDV infection.
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Optimization of Genomewide CRISPR Screens Using AsCas12a and Multi-Guide Arrays.
Genomewide loss-of-function (LOF) screening using clustered regularly interspaced short palindromic repeats (CRISPR) has facilitated the discovery of novel gene functions across diverse physiological and pathophysiological systems. A challenge with conventional genomewide CRISPR-Cas9 libraries is the unwieldy size (60,000-120,000 constructs), which is resource intensive and prohibitive in some experimental contexts. One solution to streamlining CRISPR screening is by multiplexing two or more guides per gene on a single construct, which enables functional redundancy to compensate for suboptimal gene knockout by individual guides. In this regard, AsCas12a (Cpf1) and its derivatives, for example, enhanced AsCas12a (enAsCas12a), have enabled multiplexed guide arrays to be specifically and efficiently processed for genome editing. Prior studies have established that multiplexed CRISPR-Cas12a libraries perform comparably to the larger equivalent CRISPR-Cas9 libraries, yet the most efficient CRISPR-Cas12a library design remains unresolved. In this study, we demonstrate that CRISPR-Cas12a genomewide LOF screening performed optimally with three guides arrayed per gene construct and could be adapted to robotic cell culture without noticeable differences in screen performance. Thus, the conclusions from this study provide novel insight to streamlining genomewide LOF screening using CRISPR-Cas12a and robotic cell culture.
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Genome Editing: Insights from Chemical Biology to Support Safe and Transformative Therapeutic Applications.
Programmable nuclease-based genome editing technologies, including the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system, are becoming an essential component of many applications ranging from agriculture to medicine. However, fundamental limitations currently prevent the widespread, safe, and practical use of genome editors, especially for human disease interventions. These limitations include off-target effects, a lack of control over editing activity, suboptimal DNA repair outcomes, insufficient target conversion, and inadequate delivery performance. This perspective focuses on the potential for biological chemistry to address these limitations such that newly developed genome editing technologies can enable the broadest range of potential future applications. Equally important will be the development of these powerful technologies within a relevant ethical framework that emphasizes safety and responsible innovation.
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Primary Airway Epithelial Cell Gene Editing Using CRISPR-Cas9.
The adaptation of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated endonuclease 9 (CRISPR-Cas9) machinery from prokaryotic organisms has resulted in a gene editing system that is highly versatile, easily constructed, and can be leveraged to generate human cells knocked out (KO) for a specific gene. While standard transfection techniques can be used for the introduction of CRISPR-Cas9 expression cassettes to many cell types, delivery by this method is not efficient in many primary cell types, including primary human airway epithelial cells (AECs). More efficient delivery in AECs can be achieved through lentiviral-mediated transduction, allowing the CRISPR-Cas9 system to be integrated into the genome of the cell, resulting in stable expression of the nuclease machinery and increasing editing rates. In parallel, advancements have been made in the culture, expansion, selection, and differentiation of AECs, which allow the robust generation of a bulk edited AEC population from transduced cells. Applying these methods, we detail here our latest protocol to generate mucociliary epithelial cultures knocked out for a specific gene from donor-isolated primary human basal airway epithelial cells. This protocol includes methods to: (1) design and generate lentivirus which targets a specific gene for KO with CRISPR-Cas9 machinery, (2) efficiently transduce AECs, (3) culture and select for a bulk edited AEC population, (4) molecularly screen AECs for Cas9 cutting and specific sequence edits, and (5) further expand and differentiate edited cells to a mucociliary airway epithelial culture. The AEC knockouts generated using this protocol provide an excellent primary cell model system with which to characterize the function of genes involved in airway dysfunction and disease.
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Magnetogenetics with Piezo1 Mechanosensitive Ion Channel for CRISPR Gene Editing.
Regulation of genetic activity in single cells and tissues is pivotal to determine key cellular functions in current biomedicine, yet the conventional biochemical activators lack spatiotemporal precision due to the diffusion-mediated slow kinetics and nonselectivity. Here, we describe a magnetogenetic method for target-specific activation of a clustered regularly interspaced short palindromic repeats (CRISPR) system for the regulation of intracellular proteins. We used magnetomechanical force generated by the magnetic nanostructure to activate pre-encoded Piezo1, the mechanosensitive ion channel, on the target cell. The activated Piezo1 further triggers the intracellular Ca2+ signaling pathway, inducing the pre-encoded genes to express genes of interest (GOIs), which is Cas9 protein for the CRISPR regulation of the target proteins. We demonstrated that this magnetogenetic CRISPR system successfully edits the target genome for both in vitro and pseudo-in vivo environments, providing a versatile magnetic platform for remote gene editing of animals with various size scales.
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TALEN- and CRISPR-enhanced DNA homologous recombination for gene editing in zebrafish.
The TALE nuclease and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas) have been developed as important tools for genome editing in zebrafish. Here we describe a CRISPR/Cas9-based approach for generating site-specific gene targeting in zebrafish using DNA double-strand breaks induced homologous recombination (HR)-dependent repair mechanism. Through comicroinjection of Cas9 mRNA, guide RNA targeting genomic DNA sequence corresponding to the twist2 gene and the corresponding double-strand long arm donor template with a point mutation identified in human, HR-mediated knock-in of the expected targeting sequence was obtained. To facilitate identification of germline transmission of targeted mutation, a method of screening sperms of male founder fish is designed.
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