Eukaryotes
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1307608 | ENA |
REPOSITORIES: ENA
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Project description:The therapeutic application of base editors is currently limited by their large sizes, which are often beyond the packaging capabilities of adeno-associated viral (AAV) vectors. Despite recent progress in mega genome mining that has identified a diverse array of compact CRISPR proteins (e.g. Cas12f, TnpB, and IscB), the resulting miniature base editors are often exhibited reduced activities and a limited targeting scope, leaving a broad spectrum of disease-relevant genetic variants inaccessible. In this study, we have developed a platform, designated as Zinc Finger Proteins (ZFP)-enhanced miniature base editor (zmBE), which integrates programmable DNA-binding domains to enhance the efficiency and expand targeting scope of miniature base editors, including those based on Un1Cas12f1 and OgeuIscB. Utilizing protein language model (PLM) for the design of ZFPs further simplified development and optimization of zmBEs tailored to a specific target. Leveraging these methodologies, we engineered a zmBE that effectively induced the SMN2 exon 7 A6>G conversion and restored SMN2 exon 7 inclusion. Our study thus provides a versatile platform for developing miniature base editors for in vivo therapeutic applications.
2025-04-18 | GSE294498 | GEO
Project description:Cancer cell proliferation requires precise control of E2F1 activity; excess activity promotes apoptosis. Here, we developed cell-permeable and bioavailable macrocycles that selectively kill small cell lung cancer (SCLC) cells with inherent high E2F1 activity by blocking RxL-mediated interactions of cyclin A and cyclin B with select substrates. Genome-wide CRISPR/Cas9 knockout and random mutagenesis screens found that cyclin A/B RxL macrocyclic inhibitors (cyclin A/Bi) induced apoptosis paradoxically by cyclin B- and Cdk2-dependent spindle assembly checkpoint activation (SAC). Mechanistically, cyclin A/Bi hyperactivate E2F1 and cyclin B by blocking their RxL-interactions with cyclin A and Myt1, respectively, ultimately leading to SAC activation and mitotic cell death. Base editor screens identified cyclin B variants that confer cyclin A/Bi resistance including several variants that disrupted cyclin B:Cdk interactions. Unexpectedly but consistent with our base editor and knockout screens, cyclin A/Bi induced the formation of neo-morphic Cdk2-cyclin B complexes that promote SAC activation and apoptosis. Finally, orally-bioavailable cyclin A/Bi robustly inhibited tumor growth in chemotherapy-resistant patient-derived xenograft models of SCLC. This work uncovers gain-of-function mechanisms by which cyclin A/Bi induce apoptosis in cancers with high E2F activity, and suggests cyclin A/Bi as a therapeutic strategy for SCLC and other cancers driven by high E2F activity.
2025-05-30 | PXD055112 | Pride
Project description:The most common form of genetic heart disease is hypertrophic cardiomyopathy (HCM), which is caused by mutations in cardiac sarcomeric genes and leads to abnormal heart muscle thickening. Complications of HCM include heart failure, arrhythmia, and sudden cardiac death. The dominant-negative c.1208 G>A (p.R403Q) mutation in b-myosin (MYH7) is a common and well-studied mutation that leads to increased cardiac contractility and HCM onset. Here we identify an adenine base editor (ABE) and single-guide RNA system that can efficiently correct this human pathogenic mutation with minimal off-target and bystander editing. We show that delivery of base editing components rescues pathological manifestations of HCM in iPSC-cardiomyocytes derived from HCM patients and in a humanized mouse model of HCM. Our findings demonstrate the use of base editing to treat inherited cardiac diseases and prompt the further development of ABE-based therapies to correct a variety of monogenic mutations causing cardiac disease.
2022-10-25 | GSE201755 | GEO
Project description:Current base editors use DNA deaminases, including cytidine deaminase in cytidine base editor (CBE) or adenine deaminase in adenine base editor (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for base editors capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel base editors, but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.
2024-01-23 | GSE253655 | GEO
Project description:Prion disease is caused by misfolding of the prion protein (PrP) into pathogenic self-propagating conformations, leading to rapid onset dementia and death. However, elimination of endogenous PrP can halt prion disease progression. Here, we describe CHARM, a compact, enzyme-free epigenetic editor capable of silencing transcription through programmable targeted DNA methylation. Using a histone H3 tail-Dnmt3l fusion, CHARM recruits and activates the endogenous DNA methyltransferases, thereby reducing transgene size and bystander effects. When delivered to the mouse brain by an adeno-associated viral (AAV) vector, PRNP-targeted CHARM ablates PrP expression across the brain. We temporally limit editor expression by implementing a kinetically-tuned self-silencing approach. CHARM represents a broadly applicable strategy to programmably prevent expression of pathogenic proteins, including those implicated in other neurodegenerative diseases.
2024-05-03 | GSE255987 | GEO
Project description:Base editor mediated correction of a tau mutation rescues cognitive decline in a mouse model of Alzheimers disease
| PRJNA909014 | ENA
Project description:Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show Efficacy of cytosine and adenine base editors in modelingCTNNB1hot-spot mutations in hepatocyte organoids. Next, we use C>T base editors to insert nonsense mutations inPTENin endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug screening assays on organoids harboring eitherPTENorPTENandPIK3CAmutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C>T and A>G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.
2023-07-30 | GSE236490 | GEO
Project description:We show that delivering the mitochondrial base editor DdCBEs via AAV transduction of somatic cells efficiently produces precise base editing of the intended region.
2022-01-19 | GSE184064 | GEO