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G-quadruplex-guided RNA engineering to modulate CRISPR-based genomic regulation.
It is important to develop small moelcule-based methods to modulate gene editing and expression in human cells. The roles of the G-quadruplex (G4) in biological systems have been widely studied. Here, G4-guided RNA engineering is performed to generate guide RNA with G4-forming units (G4-gRNA). We further demonstrate that chemical targeting of G4-gRNAs holds promise as a general approach for modulating gene editing and expression in human cells. The rich structural diversity of RNAs offers a reservoir of targets for small molecules to bind, thus creating the potential to modulate RNA biology.
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Conserved Opposite Functions in Plant Resistance to Biotrophic and Necrotrophic Pathogens of the Immune Regulator SRFR1.
Plant immunity is mediated in large part by specific interactions between a host resistance protein and a pathogen effector protein, named effector-triggered immunity (ETI). ETI needs to be tightly controlled both positively and negatively to enable normal plant growth because constitutively activated defense responses are detrimental to the host. In previous work, we reported that mutations in SUPPRESSOR OF rps4-RLD1 (SRFR1), identified in a suppressor screen, reactivated EDS1-dependent ETI to Pseudomonas syringae pv. tomato (Pto) DC3000. Besides, mutations in SRFR1 boosted defense responses to the generalist chewing insect Spodoptera exigua and the sugar beet cyst nematode Heterodera schachtii. Here, we show that mutations in SRFR1 enhance susceptibility to the fungal necrotrophs Fusarium oxysporum f. sp. lycopersici (FOL) and Botrytis cinerea in Arabidopsis. To translate knowledge obtained in AtSRFR1 research to crops, we generated SlSRFR1 alleles in tomato using a CRISPR/Cas9 system. Interestingly, slsrfr1 mutants increased expression of SA-pathway defense genes and enhanced resistance to Pto DC3000. In contrast, slsrfr1 mutants elevated susceptibility to FOL. Together, these data suggest that SRFR1 is functionally conserved in both Arabidopsis and tomato and functions antagonistically as a negative regulator to (hemi-) biotrophic pathogens and a positive regulator to necrotrophic pathogens.
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The dawn of the CRISPR/Cas9 gene therapy era.
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CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells.
CRISPR genome engineering has become a powerful tool to functionally investigate the complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity, the utility of current approaches is restricted by poor knockout efficiencies or limited specificity for mature cell lineages in vivo. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of mature primary mouse innate lymphocyte cells (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naive natural killer (NK) cells and bone-marrow-derived conventional dendritic cell precursors (cDCPs). This resource will enhance target gene discovery and offer a specific and simplified approach to gene editing in the mouse innate immune system.
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Myospreader improves gene editing in skeletal muscle by myonuclear propagation.
Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.
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Improving environmental stress resilience in crops by genome editing: insights from extremophile plants.
In basic and applied sciences, genome editing has become an indispensable tool, especially the versatile and adaptable CRISPR/Cas9 system. Using CRISPR/Cas9 in plants has enabled modifications of many valuable traits, including environmental stress tolerance, an essential aspect when it comes to ensuring food security under climate change pressure. The CRISPR toolbox enables faster and more precise plant breeding by facilitating: multiplex gene editing, gene pyramiding, and de novo domestication. In this paper, we discuss the most recent advances in CRISPR/Cas9 and alternative CRISPR-based systems, along with the technical challenges that remain to be overcome. A revision of the latest proof-of-concept and functional characterization studies has indeed provided more insight into the quantitative traits affecting crop yield and stress tolerance. Additionally, we focus on the applications of CRISPR/Cas9 technology in regard to extremophile plants, due to their significance on: industrial, ecological and economic levels. These still unexplored genetic resources could provide the means to harden our crops against the threat of climate change, thus ensuring food security over the next century.
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Generation of Cellular Biofactories for the Scalable Production of Surface-Engineered Extracellular Vesicles via CRISPR Genome Editing.
Extracellular vesicles (EVs) are versatile biological nanoparticles with applications in therapeutics, diagnostics, and biotechnology. Current production methods relying on transient transfection or chemical conjugation suffer from high variability, limited scalability, and heterogeneous EV populations. Here, we present a synthetic-biology-based biomaterial manufacturing platform that uses CRISPR-Cas9 genome editing to generate stable HEK293T cell lines for continuous production of surface-functionalized EVs. A fusion construct encoding mCherry-C1C2 was site-specifically integrated into the AAVS1 safe-harbor locus, enabling consistent and heritable expression of EV membrane proteins without repeated transfection. Engineered cells produced EVs with uniform size (120-130 nm), preserved canonical markers (CD63 and ALIX), and enhanced surface-display efficiency compared with transiently transfected controls. These vesicles exhibited robust cellular uptake and maintained structural and functional stability for over 25 passages (∼3 months), confirming durable genome-encoded production. Overall, this platform eliminates batch-to-batch variability inherent to transient systems and provides a genetically defined route to biofunctional nanomaterial fabrication. This approach links genetic design to nanoscale surface functionality, establishing a versatile foundation for reproducible biomanufacturing of engineered EVs for biomaterial, therapeutic, and diagnostic applications.
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Beyond Permanent Genome Editing: Molecular Delivery Strategies for RNA Editing and Epigenome-Editing Therapeutics.
Reversible genetic medicines are emerging as controllable alternatives to permanent genome editing by enabling programmable modulation of RNA sequence, transcript abundance, chromatin state, and gene expression without irreversible genomic alteration. However, reversibility is not a single binary property: transient editor exposure, decay of the molecular effect, recovery of cellular function, and clinical capacity to stop, redose, or counteract treatment may diverge. This review therefore distinguishes mechanistic, functional, and clinical reversibility while examining targeted delivery systems for RNA-editing and epigenome-editing therapeutics. Key payloads include ADAR-recruiting oligonucleotides, CRISPR-Cas13 RNA editors, guide RNAs, chemically modified RNAs, editor-encoding mRNAs, dCas9 transcriptional regulators, DNA methylation editors, histone-modifying systems, and CRISPRoff-like platforms. We evaluate extracellular and intracellular delivery barriers, including nuclease degradation, immune recognition, renal clearance, liver uptake, cellular entry, endosomal escape, cytoplasmic release, nuclear localization, chromatin access, editing-window duration, off-target activity, immunogenicity, repeat-dosing feasibility, manufacturing, quality control, potency assays, and regulatory translation. Overall, delivery systems for reversible genetic medicines should be judged by tissue selectivity, functional editing, duration of action, reversibility after treatment withdrawal, safety, manufacturability, and clinical controllability.
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New advancements in CRISPR based gene therapy of Duchenne muscular dystrophy.
Duchenne muscular dystrophy (DMD) is caused by the dystrophin gene mutations and is one of the most common and lethal human hereditary disorders. A novel therapeutic approach using CRISPR technology has gained attention in the treatment of DMD. Gene replacement strategies are being proposed as a promising therapeutic option to compensate the loss of function mutations. Although, the large size of the dystrophin gene and the limitations of the existing gene replacement approach, could mean the gene delivery of shortened versions of dystrophin such as midystrophin and microdystrophins. There are also other approaches: including Targeted removal of dystrophin exons to restore the reading-frame; Dual sgRNA-directed DMD exon deletion, CRISPR-SKIP strategy; reframing of dystrophin using Prime Editing technology; exon removal using twin prime technology; TransCRISTI technology to targeted exon integration into dystrophin gene. Here we provide an overview of recent progresses in dystrophin gene editing using updated versions of CRISPR to introduce novel opportunities in DMD gene therapy. Overall, the novel CRISPR based technologies are improving and expanding to allow the application of more precise gene editing for the treatment of DMD.
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Assessing gene function in human B cells: CRISPR/Cas9-based gene editing and mRNA-based gene expression in healthy and tumor cells.
Robust methods for manipulation of human B cells, isolated from healthy donors and patients with B cell disorders, has the potential to significantly accelerate B cell research. Our work describes a step-by-step protocol to perform electroporation-based screening of gene function in B cells through the use of Cas9 ribonuclecomplexes and in vitro produced mRNA.
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