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Photoactivatable nanoCRISPR/Cas9 System Based on crRNA Reversibly Immobilized on Carbon Nanoparticles.
Here, we proposed a new approach to engineering a photoactivatable CRISPR/Cas9 gene-editing system. The novel nanoCRISPR/Cas9 system is based on the use of auxiliary photocleavable oligodeoxyribonucleotides (PC-DNAs) complementary to crRNA. PC-DNAs contained up to three UV-sensitive linkers made of 1-(2-nitrophenyl)-1,2-ethanediol inside the oligonucleotide chain. Immobilizing PC-DNAs on the surface of carbon nanoparticles through 3'-terminal pyrene residue provided sufficient blocking of crRNA (and corresponding Cas9 activity) before UV irradiation and allows for crRNA release after UV irradiation at 365 nm, which restores Cas9 activity. We optimized the length of blocking photocleavable oligonucleotide, number of linkers, time of irradiation, and the type of carbon nanoparticles. Based on the results, we consider the nanoCRISPR/Cas9 system involving carbon-encapsulated iron nanoparticles the most promising. It provides the greatest difference of functional activity before/after irradiation and can be used in prospective for magnetic field-controlled delivery of CRISPR system into the target cells or tissues and spatiotemporal gene editing induced by UV irradiation.
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Editing the Epigenome: Overview, Open Questions, and Directions of Future Development.
The introduction of CRISPR/Cas has resulted in a strong impulse for the field of gene-targeted epigenome reprogramming. In this approach EpiEditors are applied in cells, which consist of a DNA-binding part for targeting and a functional part to induce chromatin modifications at targeted genome loci. The accumulating evidence of epigenetic reprogramming of a given genomic locus resulting in gene expression changes indicated causal relationships of epigenetic marks instructing gene expression and opened the field for mainstream applications. In this perspective, an overview of the current status of the field is provided, including its applications and future perspectives. The dependence of critical parameters like specificity, effectivity, and sustainability of epigenome editing on experimental settings and conditions including the expression levels and the duration of the expression of the EpiEditors, their DNA-binding affinity and specificity, and the cross talk between EpiEditors and cellular chromatin modifiers is discussed. Once established in fully functional "plug-and-play" mode, epigenome editing will allow to better understand epigenetic expression control and to translate such knowledge into therapeutic tools.
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CRISPR/Cas9 genome editing for comparative genetic analysis related to soy sauce brewing in Aspergillus sojae industrial strains.
Aspergillus sojae has traditionally been used in soy sauce brewing. Genetic modification techniques have been established in A. sojae, but it is difficult to apply them to various industrial strains. Although we have previously developed a CRISPR/Cpf1 system for genetic modification of A. sojae, another genome editing system was required for versatile modification. In addition, repetitive genetic modification using the CRISPR system has not been established in A. sojae. In this study, we demonstrated mutagenesis, gene deletion/integration, and large deletion of a chromosomal region in A. sojae using the CRISPR/Cas9 system. We also successfully performed repetitive genetic modification using a method that involved forced recycling of genome-editing plasmids. Moreover, we demonstrated that the effects of genetic modification related to soy sauce brewing differed among A. sojae industrial strains. These results showed that our technique of using the CRISPR/Cas9 system is a powerful tool for genetic modification in A. sojae.
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Genome editing with retroelements.
RNA-guided DNA writing enzymes offer promise for programmable gene insertion.
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PcoCas12a: A novel CRISPR enzyme from Prevotella copri enhancing TCR-T-cell tumor suppression.
Genome editing technologies have been widely utilized in cell engineering, demonstrating immense potential in cell and gene therapy. However, an optimal gene-editing enzyme for immune cell editing remains unidentified. In this study, we identified a novel gene editing enzyme, termed CRISPR/PcoCas12a, derived from Prevotella copri, which recognizes a 5'-YYN PAM sequence. We demonstrated that CRISPR/PcoCas12a offers a broader range of editing sites and superior editing efficiency at specific loci compared to AsCas12a. Furthermore, we illustrated its capability to enhance tumor suppression by targeting DGKα in TCR-T cells. DGKα functions as a negative regulator of T cell function, and its knockout significantly boosts the antitumor efficacy of TCR-T cells. The knockout efficiency and tumor suppressor ability of PcoCas12a targeting DGKα were markedly higher than those achieved with AsCas12a. Single-cell sequencing data confirmed that PcoCas12a-mediated DGKα gene knockout improves the tumor suppressive capabilities of T cells by promoting T-cell activation and strengthening immune regulatory responses. These findings establish PcoCas12a as a highly efficient enzyme for T cell editing, indicating its potential application in T-cell therapy.
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Harnessing snakehead rhabdovirus (SHRV) for gene editing by installment of CRISPR/Cas9 in viral genome.
As there is no risk of viral genome integration into host chromosome, cytoplasmic RNA viruses can be a safer vehicle to deliver CRISPR/Cas system. Snakehead rhabdovirus (SHRV) is a piscine RNA virus belonging to the family Rhabdoviridae, and, in the present study, we evaluated the availability of SHRV as a tool for CRISPR/Cas9 delivery in mammalian cells. SHRV was grown well in baby hamster kidney (BHK-21) cells at 28 °C, and the replication ability was greatly reduced by temperature up-shift to 37 °C. We rescued a recombinant SHRV that harboring not only the interferon regulatory factor 9 (IRF9) gene-targeting single-guide RNA (sgRNA) but also Cas9 gene in the genome using the reverse genetic technology. The IRF9 gene of BHK-21 cells was knocked-out by the infection with the IRF9 gene-targeting rSHRV. Moreover, the rSHRVs were sharply disappeared in the cells by elevating temperature to 37 °C, suggesting the possible regulation of knockout efficiency before virus infection-caused cell damage. Although further optimization researches are needed to enhance the editing efficiency using the recombinant SHRV, to our knowledge, this is the first report on the possible applicability of piscine RNA virus for the gene editing in mammalian cells.
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Optimizing Agrobacterium-Mediated Transformation and CRISPR-Cas9 Gene Editing in the tropical japonica Rice Variety Presidio.
Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium-mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knockout mutations in the phytoene desaturase (PDS) and young seedling albino (YSA) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties.
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The Future of Gene Editing - Toward Scientific and Social Consensus.
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CRISPR-cas9-Mediated Gene Editing to Reverse Oncogenic Mutations in Oral Squamous Cell Carcinoma.
INTRODUCTION: Oral squamous cell carcinoma (OSCC) is a genetically driven malignancy characterized by a high burden of oncogenic mutations that contribute to aggressive tumor behavior, therapeutic resistance, and poor survival outcomes. Conventional treatment modalities largely target downstream molecular pathways without correcting the underlying genetic aberrations, underscoring the need for precision-based therapeutic strategies. This study aimed to assess the feasibility and functional impact of CRISPR-Cas9-mediated gene editing in reversing oncogenic mutations associated with OSCC using an in vitro experimental model. MATERIALS AND METHODS: A controlled in vitro experimental study was conducted using the established human OSCC cell lines harboring mutations in key oncogenic genes. Cells were divided into control, mock-transfected, and CRISPR-Cas9-edited groups. Target-specific single-guide RNAs were designed to correct oncogenic mutations using advanced CRISPR-based editing platforms. Gene-editing efficiency was validated by molecular assays, while functional outcomes were assessed using cell proliferation analysis. Statistical evaluation was done using the one-way ANOVA with significance set at P < 0.05. RESULTS: CRISPR-Cas9-edited OSCC cells demonstrated a significant reduction in cell proliferation compared to control and mock-transfected groups ( P < 0.001). No significant variation was found between control and mock-transfected cells, confirming minimal procedural influence. CONCLUSION: CRISPR-Cas9-mediated correction of oncogenic mutations effectively suppresses malignant cellular proliferation in OSCC, highlighting its promising role as a precision therapeutic strategy in oral cancer management.
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Advances in CRISPR/Cas9-based research related to soybean [Glycine max (Linn.) Merr] molecular breeding.
Soybean [Glycine max (Linn.) Merr] is a source of plant-based proteins and an essential oilseed crop and industrial raw material. The increase in the demand for soybeans due to societal changes has coincided with the increase in the breeding of soybean varieties with enhanced traits. Earlier gene editing technologies involved zinc finger nucleases and transcription activator-like effector nucleases, but the third-generation gene editing technology uses clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The rapid development of CRISPR/Cas9 technology has made it one of the most effective, straightforward, affordable, and user-friendly technologies for targeted gene editing. This review summarizes the application of CRISPR/Cas9 technology in soybean molecular breeding. More specifically, it provides an overview of the genes that have been targeted, the type of editing that occurs, the mechanism of action, and the efficiency of gene editing. Furthermore, suggestions for enhancing and accelerating the molecular breeding of novel soybean varieties with ideal traits (e.g., high yield, high quality, and durable disease resistance) are included.
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