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Diagnostic applications and therapeutic option of Cascade CRISPR/Cas in the modulation of miRNA in diverse cancers: promises and obstacles.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology is a molecular tool specific to sequences for engineering genomes. Among diverse clusters of Cas proteins, the class 2/type II CRISPR/Cas9 system, despite several challenges, such as off-target effects, editing efficiency, and efficient delivery, has shown great promise for driver gene mutation discovery, high-throughput gene screening, epigenetic modulation, nucleic acid detection, disease modeling, and more importantly for therapeutic purposes. CRISPR-based clinical and experimental methods have applications across a wide range of areas, especially for cancer research and, possibly, anticancer therapy. On the other hand, given the influential role of microRNAs (miRNAs) in the regulations of cellular division, carcinogenicity, tumorigenesis, migration/invasion, and angiogenesis in diverse normal and pathogenic cellular processes, in different stages of cancer, miRNAs are either oncogenes or tumor suppressors, according to what type of cancer they are involved in. Hence, these noncoding RNA molecules are conceivable biomarkers for diagnosis and therapeutic targets. Moreover, they are suggested to be adequate predictors for cancer prediction. Conclusive evidence proves that CRISPR/Cas system can be applied to target small non-coding RNAs. However, the majority of studies have highlighted the application of the CRISPR/Cas system for targeting protein-coding regions. In this review, we specifically discuss diverse applications of CRISPR-based tools for probing miRNA gene function and miRNA-based therapeutic involvement in different types of cancers.
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Improving Gene Editing Outcomes in Human Hematopoietic Stem and Progenitor Cells by Temporal Control of DNA Repair.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas9)-mediated gene editing of human hematopoietic stem cells (hHSCs) is a promising strategy for the treatment of genetic blood diseases through site-specific correction of identified causal mutations. However, clinical translation is hindered by low ratio of precise gene modification using the corrective donor template (homology-directed repair, HDR) to gene disruption (nonhomologous end joining, NHEJ) in hHSCs. By using a modified version of Cas9 with reduced nuclease activity in G1 phase of cell cycle when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieved a four-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement after xenotransplantation of edited hHSCs into immunodeficient mice. This strategy for improving gene editing outcomes in hHSCs has important implications for the field of gene therapy, and can be applied to diseases where increased HDR/NHEJ ratio is critical for therapeutic success. Stem Cells 2019;37:284-294.
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Genome editing of PD-L1 mediated by nucleobase-modified polyamidoamine for cancer immunotherapy.
Immune checkpoint blockade therapy against programmed death protein-1 and its ligand (PD-1/PD-L1) has been accepted as a promising approach to activate the immune system's anti-tumor response. Although small interfering RNA (siRNA) or antibodies can block the PD-1/PD-L1 pathway, the effect of this blockade is temporary and reversible. Here, we developed a nano-delivery system to achieve permanent disruption of the PD-L1 gene based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) gene editing technology. In this system, the CRISPR/Cas9 plasmid was delivered into melanoma B16F10 cells using a nucleobase-modified polyamidoamine (PAMAM) derivative namely AP-PAMAM, which was constructed through the modification with 2-amino-6-chloropurine. Meanwhile, the carrier could efficiently facilitate the endosomal escape of CRISPR/Cas9 plasmid and thereby inhibit PD-L1 expression in cancer cells. Moreover, the intravenous injection of AP-PAMAM/plasmid nanoparticles could recruit and activate CD8+ T cells at the tumor site, promoting the secretion of cytokines and the killing of tumor cells. Overall, this nano-delivery system for genome editing provided a promising strategy to block the PD-1/PD-L1 pathway and obtain effective tumor immunotherapy.
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From bench to bedside: Developing CRISPR/Cas-based therapy for ocular diseases.
Vision-threatening disorders, including both hereditary and multifactorial ocular diseases, necessitate innovative therapeutic approaches. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has emerged as a promising tool for treating ocular diseases through gene editing and expression regulation. This system has contributed to the development of representative disease models, including animal models, organoids, and cell lines, thereby facilitating investigations into the pathogenesis of disease-related genes. Besides, therapeutic applications of CRISPR/Cas have been extensively explored in preclinical in vitro and in vivo studies, targeting various ocular conditions, such as retinitis pigmentosa, Leber congenital amaurosis, Usher syndrome, fundus neovascular diseases, glaucoma, and corneal diseases. Recent advancements have demonstrated the technology's potential to restore cellular homeostasis and alleviate disease phenotypes, thereby prompting a variety of clinical trials. To date, active trials include treatments for primary open angle glaucoma with MYOC mutations, refractory herpetic viral keratitis, CEP290-associated inherited retinal degenerations, neovascular age-related macular degeneration, and retinitis pigmentosa with RHO mutations. However, challenges remain, primarily concerning off-target effects, immunogenicity, ethical considerations, and regulatory particularity. To reach higher safety and efficiency before truly transitioning from bench to bedside, future research should concentrate on improving the specificity and efficacy of Cas proteins, optimizing delivery vectors, and broadening the applicability of therapeutic targets. This review summarizes the application strategies and delivery methods of CRISPR/Cas, discusses recent progress in CRISPR/Cas-based disease models and therapies, and provides an overview of the landscape of clinical trials. Current obstacles and future directions regarding the bench-to-bedside transition are also discussed.
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Generating genetically modified mice using CRISPR/Cas-mediated genome engineering.
Mice with specific gene modifications are valuable tools for studying development and disease. Traditional gene targeting in mice using embryonic stem (ES) cells, although suitable for generating sophisticated genetic modifications in endogenous genes, is complex and time-consuming. We have recently described CRISPR/Cas-mediated genome engineering for the generation of mice carrying mutations in multiple genes, endogenous reporters, conditional alleles or defined deletions. Here we provide a detailed protocol for embryo manipulation by piezo-driven injection of nucleic acids into the cytoplasm to create gene-modified mice. Beginning with target design, the generation of gene-modified mice can be achieved in as little as 4 weeks. We also describe the application of the CRISPR/Cas technology for the simultaneous editing of multiple genes (five genes or more) after a single transfection of ES cells. The principles described in this protocol have already been applied in rats and primates, and they are applicable to sophisticated genome engineering in species in which ES cells are not available.
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Genome engineering of Clostridium difficile using the CRISPR-Cas9 system.
OBJECTIVES: Clostridium difficile is a notorious pathogenic species that can cause severe gastrointestinal infections in humans and animals. C. difficile infection (CDI) results in thousands of deaths worldwide every year. The elucidation of related mechanisms of CDI and exploration of potential therapeutic strategies are largely delayed due to the lack of efficient genetic engineering tools for C. difficile strains. METHODS: Plasmids carrying the CRISPR-Cas9 system were constructed and transformed into C. difficile through conjugation. Mutants were identified using colony PCR with primers annealing to the regions flanking the target gene deletion/integration locus. Heat-survival assay was used to compare the sporulation frequency between the mutant with spo0A deletion and the wild type strain. The fluorescence in the mutant with the insertion of the green fluorescent protein (GFP) gene was inspected under a fluorescent microscope. RESULTS: An efficient genome editing tool was developed for C. difficile based on the CRISPR-Cas9 system. With this tool, spo0A was deleted with a 100% mutation efficiency. Conversely, an anaerobic GFP gene was successfully inserted into the C. difficile chromosome (with a mutation efficiency of 80%). CONCLUSIONS: The developed CRISPR-Cas9-based genome engineering tool will facilitate functional genomic studies in C. difficile as well as the elucidation of mechanisms related to host-bacteria interaction and pathogenesis of CDI. This will be highly beneficial for the development of innovative strategies for CDI diagnostics and therapies.
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CRISPR/Cas9-mediated knockout of STAT1 in porcine-derived cell lines to elucidate the role of STAT1 in autophagy following classical swine fever virus infection.
Signal transducer and activator of transcription 1 (STAT1) plays a critical role in immune response, human STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity. Classical swine fever virus (CSFV)-infected induce autophagy, leading to immune evasion. However, there are limited reports on the function of porcine STAT1 in autophagy during CSFV infection. There is also lack of suitable in vitro models for studying porcine STAT1. The objective of this study was to establish porcine PK-15 STAT1-/- and 3D4/21 STAT1-/- cell lines using the CRISPR/Cas9 system to investigate the function of the STAT1 in autophagy. The PK-15STAT1-/- and 3D4/21STAT1-/- cell lines, featuring homozygous knockout of STAT1 gene were successfully constructed using the CRISPR/Cas9 editing system. The knockout efficiency determined to be 82.4% and 81.1%, respectively. Infection with CSFV in porcine PK-15STAT1-/- and 3D4/21STAT1-/- cells led to an observable increase in autophagosomes as evidenced by transmission electron microscope. Additionally, STAT1 knockout (STAT1-/-) by the CRISPR/Cas9 system upregulated the expression of ULK1, Beclin1, and LC3 genes, thereby enhancing autophagy during CSFV infection. Conversely, overexpression of STAT1 downregulated the expression of ULK1, Beclin1, and LC3 genes, leading to inhibition of autophagy during CSFV infection.The application of an autophagy dual-fluorescent-tracking plasmid demonstrated that STAT1 knockout enhanced autophagy accumulation during CSFV infection, while STAT1 overexpression inhibited it. Moreover, the 3D4/21STAT1-/- cell line proved to be a more suitable in vitro model compared to the PK-15STAT1-/- cell line for elucidating the involvement of STAT1 in autophagy during CSFV infection.
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Engineering a High-Fidelity MAD7 Variant with Enhanced Specificity for Precision Genome Editing via CcdB-Based Bacterial Screening.
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) nucleases enable precise genome editing, but off-target cleavage remains a critical challenge. Here, we report the development of MAD7_HF, a high-fidelity variant of the MAD7 nuclease engineered through a bacterial screening system leveraging the DNA gyrase-targeting toxic gene ccdB. This system couples survival to efficient on-target cleavage and minimal off-target activity, mimicking the transient action required for high-precision editing. Through iterative selection and sequencing validation, we identified MAD7_HF, harboring three substitutions (R187C, S350T, K1019N) that enhanced discrimination between on- and off-target sites. In Escherichia coli assays, MAD7_HF exhibited a >20-fold reduction in off-target cleavage across multiple mismatch contexts while maintaining on-target efficiency comparable to wild-type MAD7. Structural modeling revealed that these mutations stabilize the guide RNA-DNA hybrid at on-target sites and weaken interactions with mismatched sequences. This work establishes a high-throughput bacterial screening strategy that allows the identification of Cas12a variants with improved specificity at a given target site, providing a useful framework for future efforts to develop precision genome-editing tools.
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CRISPR/Cas9-based genome editing in mice by single plasmid injection.
CRISPR/Cas-mediated genome modification has opened a new era for elucidating gene function. Gene knockout mice can be generated by injecting humanized Cas9 (hCas9) mRNA and guide RNA (sgRNA) into fertilized eggs. However, delivery of RNA instead of DNA to the fertilized oocyte requires extra preparation and extra care with storage. To simplify the method of delivery, we injected the circular pX330 plasmids expressing both hCas9 and sgRNA and found that mutant mice were generated as efficiently as with RNA injection. Different from the linearized plasmid, the circular plasmid decreased the chance of integration into the host genome. We also developed the pCAG-EGxxFP reporter plasmid for evaluating the sgRNA activity by observing EGFP fluorescence in HEK293T cells. The combination of these techniques allowed us to develop a rapid, easy, and reproducible strategy for targeted mutagenesis in living mice. This chapter provides an experimental protocol for the design of sgRNAs, the construction of pX330-sgRNA and pCAG-EGxxFP-target plasmids, the validation of cleavage efficiency in vitro, and the generation of targeted gene mutant mice. These mice can be generated within a month.
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CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages.
Bacteriophages are now widely recognized as major players in a wide variety of ecosystems. Novel genes are often identified in newly isolated phages as well as in environmental metavirome studies. Most of these novel viral genes have unknown functions but appear to be coding for small, non-structural proteins. To understand their biological role, very efficient genetic tools are required to modify them, especially in the genome of virulent phages. We first show that specific point mutations and large deletions can be engineered in the genome of the virulent phage 2972 using the Streptococcus thermophilus CRISPR-Cas Type II-A system as a selective pressure to increase recombination efficiencies. Of significance, all the plaques tested contained recombinant phages with the desired mutation. Furthermore, we show that the CRISPR-Cas engineering system can be used to efficiently introduce a functional methyltransferase gene into a virulent phage genome. Finally, synthetic CRISPR bacteriophage insensitive mutants were constructed by cloning a spacer-repeat unit in a low-copy vector illustrating the possibility to target multiple regions of the phage genome. Taken together, this data shows that the CRISPR-Cas system is an efficient and adaptable tool for editing the otherwise intractable genomes of virulent phages and to better understand phage-host interactions.
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