Continuous evolution of compact protein degradation tags regulated by selective molecular glues

Science, Год журнала: 2024, Номер 383(6688)

Опубликована: Март 14, 2024

Conditional protein degradation tags (degrons) are usually >100 amino acids long or triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame SD40 using prime editing degraded otherwise inert PT-179. Cryo-electron microscopy structures ligand-bound revealed mechanistic insights into basis SD40's activity specificity. Our efforts establish system provide ZF overcome shortcomings associated existing degrons.

Язык: Английский

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Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells

Nature Biomedical Engineering, Год журнала: 2024, Номер unknown

Опубликована: Июль 10, 2024

Abstract Prime editing (PE) enables precise and versatile genome without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) F508del, a three-nucleotide deletion that is predominant cause CF. By combining six efficiency optimizations for PE—engineered guide RNAs, PEmax architecture, transient expression dominant-negative mismatch repair protein, strategic silent edits, PE6 variants proximal ‘dead’ single-guide RNAs—we increased correction efficiencies F508del from less than 0.5% in HEK293T cells 58% immortalized bronchial epithelial (a 140-fold improvement) 25% patient-derived airway cells. The also resulted minimal off-target editing, edit-to-indel ratios 3.5-fold greater those achieved by nuclease-mediated homology-directed repair, functional restoration ion channels over 50% wild-type levels (similar via combination treatment with elexacaftor, tezacaftor ivacaftor) primary Our findings support feasibility durable one-time

Язык: Английский

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Precise deletion, replacement and inversion of large DNA fragments in plants using dual prime editing

Nature Plants, Год журнала: 2025, Номер unknown

Опубликована: Янв. 13, 2025

Язык: Английский

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Beyond the promise: evaluating and mitigating off-target effects in CRISPR gene editing for safer therapeutics

Frontiers in Bioengineering and Biotechnology, Год журнала: 2024, Номер 11

Опубликована: Янв. 18, 2024

Over the last decade, CRISPR has revolutionized drug development due to its potential cure genetic diseases that currently do not have any treatment. was adapted from bacteria for gene editing in human cells 2012 and, remarkably, only 11 years later seen it’s very first approval as a medicine treatment of sickle cell disease and transfusion-dependent beta-thalassemia. However, application systems is associated with unintended off-target on-target alterations (including small indels, structural variations such translocations, inversions large deletions), which are source risk patients vital concern safe therapies. In recent years, wide range methods been developed detect unwanted effects CRISPR-Cas nuclease activity. this review, we summarize different assessment, discuss their strengths limitations, highlight strategies improve safety systems. Finally, relevance pre-clinical assessment therapeutics within current regulatory context.

Язык: Английский

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Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Июнь 8, 2024

DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no editor for thymine (T) currently. Here we develop two deaminase-free glycosylase-based T (gTBE) and C (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil glycosylase (UNG) variants. By several rounds structure-informed rational mutagenesis on UNG in cultured cells, obtain gTBE gCBE high activity T-to-S (i.e., T-to-C T-to-G) C-to-G conversions, respectively. Furthermore, conduct parallel comparison gTBE/gCBE those recently developed using other protein engineering strategies, find show the outperformance. Thus, provide editors, gTBEs gCBEs, corresponding variants, broadening targeting scope editors.

Язык: Английский

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Emerging Gene Therapeutics for Epidermolysis Bullosa under Development

International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(4), С. 2243 - 2243

Опубликована: Фев. 13, 2024

The monogenetic disease epidermolysis bullosa (EB) is characterised by the formation of extended blisters and lesions on patient's skin upon minimal mechanical stress. Causal for this severe condition are genetic mutations in genes, leading to functional impairment, reduction, or absence encoded protein within skin's basement membrane zone connecting epidermis underlying dermis. major burden affected families justifies development long-lasting curative therapies operating at genomic level. landscape causal EB steadily expanding due recent breakthroughs gene therapy field, providing promising outcomes patients suffering from disease. Currently, two therapeutic approaches show promise EB. clinically more advanced replacement strategy was successfully applied forms, a ground-breaking vivo product named beremagene geperpavec (B-VEC) recently approved US Food Drug Administration (FDA). In addition, continuous innovations both designer nucleases editing technologies enable efficient potentially safe repair permanent manner, inspiring researchers field define reach new milestones

Язык: Английский

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Recent Therapeutic Gene Editing Applications to Genetic Disorders

Current Issues in Molecular Biology, Год журнала: 2024, Номер 46(5), С. 4147 - 4185

Опубликована: Апрель 30, 2024

Recent years have witnessed unprecedented progress in therapeutic gene editing, revolutionizing the approach to treating genetic disorders. In this comprehensive review, we discuss progression of milestones leading emergence clustered regularly interspaced short palindromic repeats (CRISPR)-based technology as a powerful tool for precise and targeted modifications human genome. CRISPR-Cas9 nuclease, base prime editing taken center stage, demonstrating remarkable precision efficacy ex vivo genomic modifications. Enhanced delivery systems, including viral vectors nanoparticles, further improved efficiency safety advancing their clinical translatability. The exploration CRISPR-Cas systems beyond commonly used Cas9, such development Cas12 Cas13 variants, has expanded repertoire tools, enabling more intricate interventions. Outstandingly, represents significant leap forward, given its unparalleled versatility minimization off-target effects. These innovations paved way multitude previously incurable disorders, ranging from monogenic diseases complex polygenic conditions. This review highlights latest innovative studies field, emphasizing breakthrough technologies preclinical trials, applications realm medicine. However, challenges effects ethical considerations remain, necessitating continued research refine profiles frameworks.

Язык: Английский

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Harnessing the evolving CRISPR/Cas9 for precision oncology

Journal of Translational Medicine, Год журнала: 2024, Номер 22(1)

Опубликована: Авг. 8, 2024

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system, a groundbreaking innovation in genetic engineering, has revolutionized our approach to surmounting complex diseases, culminating CASGEVY™ approved for sickle cell anemia. Derived from microbial immune defense mechanism, CRISPR/Cas9, characterized as precision, maneuverability and universality gene editing, been harnessed versatile tool precisely manipulating DNA mammals. In the process of applying it practice, consecutive exploitation novel orthologs variants never ceases. It's conducive understanding essentialities particularly cancer, which is crucial diagnosis, prevention, treatment. CRISPR/Cas9 used not only investigate tumorous genes functioning but also model disparate cancers, providing valuable insights into tumor biology, resistance, evasion. Upon cancer therapy, instrumental developing individual precise therapies that can selectively activate or deactivate within cells, aiming cripple growth invasion sensitize cells treatments. Furthermore, facilitates development innovative treatments, enhancing targeting efficiency reprogrammed exemplified by advancements CAR-T regimen. Beyond potent screening susceptible genes, offering possibility intervening before initiative progresses. However, despite its vast potential, application research therapy accompanied significant efficacy, efficiency, technical, safety considerations. Escalating technology innovations are warranted address these issues. system revolutionizing treatment, opening up new avenues management cancers. integration this evolving clinical practice promises era precision oncology, with targeted, personalized, potentially curative patients.

Язык: Английский

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Programmable RNA base editing with photoactivatable CRISPR-Cas13

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Янв. 22, 2024

Abstract CRISPR-Cas13 is widely used for programmable RNA interference, imaging, and editing. In this study, we develop a light-inducible Cas13 system called paCas13 by fusing Magnet with fragment pairs. The most effective split site, N351/C350, was identified found to exhibit low background high inducibility. We observed significant light-induced perturbation of endogenous transcripts paCas13. further present base-editing system, herein the padCas13 editor, ADAR2 catalytically inactive fragments. editor enabled reversible editing under light in A-to-I C-to-U bases, targeting disease-relevant transcripts, fine-tuning mammalian cells vitro. also adjust post-translational modifications demonstrated ability activate target mouse model vivo. therefore RNA-modulating technique based on that enables RNAs be diversely manipulated vitro vivo, including through degradation base approach using can broadly applicable manipulating various disease states physiological processes, offering potential additional avenues research therapeutic development.

Язык: Английский

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Optimized dicot prime editing enables heritable desired edits in tomato and Arabidopsis

Nature Plants, Год журнала: 2024, Номер 10(10), С. 1502 - 1513

Опубликована: Сен. 6, 2024

Язык: Английский

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