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CRISPR/Cas9 to Silence Long Non-Coding RNAs.
Knockout (KO) of long non-coding RNAs (lncRNAs) enables functional characterization of this still poorly described group of transcripts. One of the most efficient and simplest methods to achieve complete KO of a lncRNA is by employing CRISPR/Cas gene editing. As most lncRNAs are not well annotated, their individual functional regions are often not defined, and the majority of the transcripts are not affected by single nucleotide mutations. Therefore, CRISPR/Cas KO is more challenging for lncRNAs as compared to KO of protein coding genes. Strategies for lncRNAs KO include complete removal of the entire gene, removal of the promoter and transcriptional start site, abolishing exon-exon junctions, or removing the transcriptional termination site. Here, we describe the methodology to perform CRISPR/Cas9 KO of lncRNAs in vitro using electroporation as the method of transfection of presynthesized single guide RNAs (sgRNAs) and Cas9 enzyme.
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The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing.
Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only a few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas allow directed exploration of new endogenous selectable markers. Since editing frequencies remain comparably low, a Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, which improved successful gene edits from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was expressed in the cytosol or targeted to the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.
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Therapeutic potential of CRISPR/Cas9 gene editing in engineered T-cell therapy.
Cancer patients have been treated with various types of therapies, including conventional strategies like chemo-, radio-, and targeted therapy, as well as immunotherapy like checkpoint inhibitors, vaccine and cell therapy etc. Among the therapeutic alternatives, T-cell therapy like CAR-T (Chimeric Antigen Receptor Engineered T cell) and TCR-T (T Cell Receptor Engineered T cell), has emerged as the most promising therapeutics due to its impressive clinical efficacy. However, there are many challenges and obstacles, such as immunosuppressive tumor microenvironment, manufacturing complexity, and poor infiltration of engrafted cells, etc still, need to be overcome for further treatment with different forms of cancer. Recently, the antitumor activities of CAR-T and TCR-T cells have shown great improvement with the utilization of CRISPR/Cas9 gene editing technology. Thus, the genome editing system could be a powerful genetic tool to use for manipulating T cells and enhancing the efficacy of cell immunotherapy. This review focuses on pros and cons of various gene delivery methods, challenges, and safety issues of CRISPR/Cas9 gene editing application in T-cell-based immunotherapy.
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Gene editing as a promising approach for respiratory diseases.
Respiratory diseases, which are leading causes of mortality and morbidity in the world, are dysfunctions of the nasopharynx, the trachea, the bronchus, the lung and the pleural cavity. Symptoms of chronic respiratory diseases, such as cough, sneezing and difficulty breathing, may seriously affect the productivity, sleep quality and physical and mental well-being of patients, and patients with acute respiratory diseases may have difficulty breathing, anoxia and even life-threatening respiratory failure. Respiratory diseases are generally heterogeneous, with multifaceted causes including smoking, ageing, air pollution, infection and gene mutations. Clinically, a single pulmonary disease can exhibit more than one phenotype or coexist with multiple organ disorders. To correct abnormal function or repair injured respiratory tissues, one of the most promising techniques is to correct mutated genes by gene editing, as some gene mutations have been clearly demonstrated to be associated with genetic or heterogeneous respiratory diseases. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are three innovative gene editing technologies developed recently. In this short review, we have summarised the structure and operating principles of the ZFNs, TALENs and CRISPR/Cas9 systems and their preclinical and clinical applications in respiratory diseases.
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Development of a CRISPR/Cas9-induced gene editing system for Pseudoalteromonas fuliginea and its applications in functional genomics.
Pseudoalteromonas has been used as a model system to study cold adaptation and is of widespread interest in biotechnology and ecology. To explore its physiological responses to extreme cold, uncover functional genes, and clarify their ecological roles, efficient genetic tools are essential. However, existing genetic manipulation methods in Pseudoalteromonas rely on traditional homology-based recombination, which is laborious and time-consuming in this bacterial system. Consequently, improving editing efficiency is crucial for advancing both basic research and applied potential. Here, we established a CRISPR/Cas9 system in Pseudoalteromonas and carried out an extensive investigation of the Type II CRISPR/Cas9 platform for gene editing in Pseudoalteromonas fuliginea, a representative species thriving in the frigid polar oceans. To validate the feasibility of the CRISPR/Cas system in P. fuliginea, multiple genes were selected as targets, and the gene editing effects were confirmed through phenotypic changes or gene expression. We have successfully achieved both gene knockouts and insertions in P. fuliginea, encompassing the deletion of genes such as fliJ, indA, and genes encoding Pf sRNAs, as well as the in vivo insertion of 3×FLAG and the gfp gene. The average CRISPR/Cas9 gene editing efficiency in P. fuliginea exceeded 70%. In summary, we developed an efficient CRISPR/Cas9-based editing system in P. fuliginea, which can be utilized to accelerate the development of Pseudoalteromonas as a model system for addressing fundamental questions related to extreme environmental adaptation and to fulfill its potential biotechnological applications.IMPORTANCEPseudoalteromonas fuliginea is a marine bacterium with great potential for ecological and biotechnological research, yet its genetic manipulation has long been a technical challenge. In this study, we developed a gene editing system based on CRISPR technology that enables efficient and precise genome modification in this organism. Using this system, we successfully deleted, inserted, and tagged multiple genes, including regulatory and non-coding elements, with high success rates. Notably, several of these genes are linked to key traits such as motility and stress response, which contribute to microbial adaptation in polar environments. This tool allows researchers to directly test gene function and study microbial adaptation in cold marine environments. The ability to perform reliable genetic edits in P. fuliginea opens new possibilities for its use as a model organism and will support future advances in microbial ecology, environmental microbiology, and marine biotechnology.
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Direct parental CRISPR gene editing in the predatory bug Orius strigicollis, a biocontrol agent against small arthropods.
BACKGROUND: The predatory flower bug Orius strigicollis serves as a valuable biocontrol agent against small arthropods; however, its effectiveness can vary, especially when population establishment fails due to low prey/pest densities. A promising approach to improve the efficacy of O. strigicollis as a biocontrol agent is through gene editing. However, as females lay their eggs in plant tissue, the conventional embryo injection approach is challenging in this species. RESULTS: In this study, we aimed to develop an efficient and practical gene editing technique for O. strigicollis using direct parental CRISPR (DIPA-CRISPR). Female bugs at various postemergence stages received Cas9 ribonucleoprotein injections, with subsequent genotyping of their offspring (G0) using PCR and a heteroduplex mobility assay. We targeted the kynurenine 3-monooxygenase gene (cinnabar), pivotal for insect ommochrome pigment biosynthesis. Through experimental optimization, we achieved a peak gene editing efficiency of 52%, i.e., 52% of G0 progeny carried gene-edited alleles when injecting 1 day postemergence. Notably, some gene-edited G0 adults exhibited a red-eye mosaic phenotype, in contrast to the black-eyed wild type. Crossing experiments confirmed the heritability of the introduced mutations in the subsequent generation (G1), enabling the establishment of a cinnabar-knockout line with bright red eyes. CONCLUSION: We demonstrate that our DIPA-CRISPR gene editing method tailored for O. strigicollis is efficient and practical. Our findings highlight the potency of DIPA-CRISPR as a tool for O. strigicollis genetic engineering and suggest broader applications for enhancing other biocontrol agents. © 2024 Society of Chemical Industry.
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Gene editing in common cardiovascular diseases.
Cardiovascular diseases are the leading cause of morbidity and mortality worldwide, highlighting the high socioeconomic impact. Current treatment strategies like compound-based drugs or surgeries are often limited. On the one hand, systemic administration of substances is frequently associated with adverse side effects; on the other hand, they typically provide only short-time effects requiring daily intake. Thus, new therapeutic approaches and concepts are urgently needed. The advent of CRISPR-Cas9 genome editing offers great promise for the correction of disease-causing hereditary mutations. As such mutations are often very rare, gene editing strategies to correct them are not broadly applicable to many patients. Notably, there is recent evidence that gene editing technology can also be deployed to disrupt common pathogenic signaling cascades in a targeted, specific, and efficient manner, which offers a more generalizable approach. However, several challenges remain to be addressed ranging from the optimization of the editing strategy itself to a suitable delivery strategy up to potential immune responses to the editing components. This review article discusses important CRISPR-Cas9-based gene editing approaches with their advantages and drawbacks and outlines opportunities in their application for treatment of cardiovascular diseases.
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Special Focus Issue Part II: Recruitment of solid lipid nanoparticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing.
Aim: The CRISPR/Cas9 system is a promising gene-editing tool for various anticancer therapies; however, development of a biocompatible, nonviral and efficient delivery of CRISPR/Cas9 expression systems remains a challenge. Materials & methods: Solid lipid nanoparticles (SLNs) were produced based on pseudo and 3D ternary plots. Obtained SLNs and their complexes with PX458 plasmid DNA were characterized and evaluated in terms of cytotoxicity and transfection efficiency. Results: SLNs were found to be nanosized, monodispersed, stable and nontoxic. Furthermore, they revealed similar transfection efficiency as the positive control. Conclusion: Overall, we have achieved a good SLN basis for CRISPR/Cas9 delivery and have the potential to produce SLNs with targeted anticancer properties by modifying production parameters and components to facilitate translating CRISPR/Cas9 into preclinical studies.
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CRISPR/Cas9: the gold standard of genome editing?
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Comparison of Prime Editing and CRISPR-Cas Mediated HDR in Correcting CFTR-F508del Mutation in Patient iPSC-Derived Airway Basal Cells.
Gene editing therapy represents a promising strategy to permanently cure cystic fibrosis (CF). This paper presents the results of correcting the most common CF mutation, F508del, in the CFTR gene in airway basal cells (BCs) obtained from patients, using both classical CRISPR-Cas9-mediated homology-directed repair (HDR) and prime editing techniques. Our findings demonstrate that both methods achieve mutation correction with efficiencies reaching up to 18.9% of alleles in successfully transfected cells. However, the CRISPR-Cas9 approach results in insertions and deletions (indels) in approximately 15% of alleles at the editing site, whereas prime editing induced only minimal indel events. Functional analysis evaluating the effectiveness of gene editing revealed that prime editing of the F508del mutation in the CFTR gene-using the pegRNA4 combined with the PE2-NG variant-restores CFTR channel conductance, comparable to triple therapy with CFTR modulators (ivacaftor/tezacaftor/elexacaftor). While both prime editing and CRISPR-HDR resulted in successful correction of the CFTR-F508del mutation in patient BCs, prime editing exhibited a low frequency of unintended modifications both within and outside the editing locus, making this approach a promising candidate for the development of gene therapy strategies for CF.
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