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Activation of the cGAS-STING pathway combined with CRISPR-Cas9 gene editing triggering long-term immunotherapy.
Effective activation of cGAS-STING pathway combined with immune checkpoint blockade (ICB) within the immunosuppressive tumor microenvironment to induce stronger immune responsiveness yet remains challenging. CRISPR-Cas9 gene editing technology, which offers the benefits of permanence and irreversibility, could recognize the target genome sequence with sgRNA (Guide RNA) and guide the Cas9 protease to knock down the target gene. Herein, a nanoplatform (HMnMPH) for dual activation of cGAS-STING pathway in combination with CRISPR-Cas9 gene editing to silence programmed death ligand 1 (PD-L1) to trigger long-term immunotherapy was reported. The HMnMPH consists of hollow manganese dioxide (HMn) loaded with STING agonist (MSA-2) and CRISPR-Cas9/sg-PD-L1 plasmid with further modification of hyaluronic acid (HA). In acidic and GSH overexpressed tumor environment, HMnPMH was degraded to release large amounts of Mn ions and STING agonists, strongly and persistently activating the cGAS-STING pathway to promote the release of type I interferon and pro-inflammatory factors. Meanwhile, the released CRISPR-Cas9 plasmid could knockdown the PD-L1 immune checkpoint and restart immunosuppressive T cells to differentiate into cytotoxic T lymphocytes significantly, which reduced the activity of primary and distal tumors and demonstrated a long-term immune memory effect on distal tumors.
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Genome editing in animals: why FDA regulation matters.
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Parents weigh promise and risks of germline editing.
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A versatile CRISPR/Cas9 system off-target prediction tool using language model.
Genome editing with the CRISPR/Cas9 system has revolutionized life and medical sciences, particularly in treating monogenic genetic diseases by enabling long-term therapeutic effects from a single intervention. However, the CRISPR/Cas9 system can tolerate mismatches and DNA/RNA bulges at target sites, leading to unintended off-target effects that pose challenges for gene-editing therapy development. Existing high-throughput detection and in silico prediction methods are often limited to specifically designed single guide RNAs (sgRNAs) and perform poorly on unseen sequences. To address these limitations, we introduce CCLMoff, a deep learning framework for off-target prediction that incorporates a pretrained RNA language model from RNAcentral. CCLMoff captures mutual sequence information between sgRNAs and target sites and is trained on a comprehensive, updated dataset. This approach enables accurate off-target identification and strong generalization across diverse NGS-based detection datasets. Model interpretation reveals the biological importance of the seed region, underscoring CCLMoff's analytical capabilities. The development of CCLMoff lays the foundation for a comprehensive, end-to-end sgRNA design platform, enhancing both the precision and efficiency of CRISPR/Cas9-based therapeutics. CCLMoff is a versatile tool and is publicly available at github.com/duwa2/CCLMoff .
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TALEDs complete the toolkit for editing human mitochondrial DNA.
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An uneven race: genome editing for parasitic worms.
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Agrobacterium tumefaciens-Mediated Genome Editing in Banana.
This protocol outlines the Agrobacterium tumefaciens-mediated transformation process for genome editing in banana (Musa spp.). As a crop of significant agricultural and economic importance globally, improving banana cultivars is crucial for addressing challenges such as disease resistance, climate resilience and yield enhancement. The procedure presented here involves the establishment of embryogenic cell suspensions (ECSs) from immature male flowers, followed by co-cultivation with Agrobacterium containing the desired gene construct. Key steps include callus induction, ECS generation and maintenance, transformation using plasmid construct containing genome editing reagents such as CRISPR/Cas9, and regeneration. The method ensures the editing of target genes in the banana genome, facilitating genetic improvements.
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Highly efficient genome editing using oocyte-specific zcas9 transgenic zebrafish.
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National Academies relax stance on germline edits.
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CRISPR/Cas9-Assisted Genome Editing in Murine Embryonic Stem Cells.
The study of gene function in normal human physiology and pathophysiology is complicated by countless factors such as genetic diversity (~98 million SNPs identified in the human genome as of June 2015), environmental exposure, epigenetic imprinting, maternal/in utero exposure, diet, exercise, age, sex, socioeconomic factors, and many other variables. Inbred mouse lines have allowed researchers to control for many of the variables that define human diversity but complicate the study of the human genome, gene/protein function, cellular and molecular pathways, and countless other genetic diseases. Furthermore, genetically modified mouse models enable us to generate and study mice whose genomes differ by as little as a single point mutation while controlling for non-genomic variables. This allows researchers to elucidate the quintessential function of a gene, which will further not only our scientific understanding, but provide key insight into human health and disease. Recent advances in CRISPR/Cas9 genome editing have revolutionized scientific protocols for introducing mutations into the mammalian genome. The ensuing chapter provides an overview of CRISPR/Cas9 genome editing in murine embryonic stem cells for the generation of genetically modified mouse models.
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