Chromatin context-dependent regulation and epigenetic manipulation of prime editing

Cell, Journal Year: 2024, Volume and Issue: 187(10), P. 2411 - 2427.e25

Published: April 11, 2024

We set out to exhaustively characterize the impact of cis-chromatin environment on prime editing, a precise genome engineering tool. Using highly sensitive method for mapping genomic locations randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% 94% an identical target site and edit. Position effects efficiency are well predicted chromatin marks, e.g., positively H3K79me2 negatively H3K9me3. Next, developed multiplex perturbational framework assess interaction trans-acting factors with outcomes. Applying this DNA repair factors, identify HLTF as context-dependent repressor editing. Finally, several lines evidence suggest that active transcriptional elongation enhances Consistent this, show can robustly decrease or increase preceding it CRISPR-mediated silencing activation, respectively.

Language: Английский

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High-throughput evaluation of genetic variants with prime editing sensor libraries

Nature Biotechnology, Journal Year: 2024, Volume and Issue: unknown

Published: March 12, 2024

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install evaluate genetic variants, but previous approaches have limited by the variable efficiency prime guide RNAs. Here we present high-throughput sensor strategy couples RNAs with synthetic versions their cognate target sites quantitatively assess functional impact endogenous variants. We screen over 1,000 cancer-associated variants TP53-the most frequently mutated gene in cancer-to identify alleles p53 function mechanistically diverse ways. find certain TP53 particularly those oligomerization domain, display opposite phenotypes exogenous overexpression systems. Our results emphasize physiological importance dosage shaping native stoichiometry protein-protein interactions, establish framework for studying sequence context at scale.

Language: Английский

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Precise genome-editing in human diseases: mechanisms, strategies and applications

Signal Transduction and Targeted Therapy, Journal Year: 2024, Volume and Issue: 9(1)

Published: Feb. 26, 2024

Precise genome-editing platforms are versatile tools for generating specific, site-directed DNA insertions, deletions, and substitutions. The continuous enhancement of these has led to a revolution in the life sciences, which promises deliver novel therapies genetic disease. can be traced back 1950s with discovery DNA's double-helix and, after 70 years development, evolved from crude vitro applications wide range sophisticated capabilities, including vivo applications. Nonetheless, precise faces constraints such as modest efficiency, delivery challenges, off-target effects. In this review, we explore genome-editing, focus on introduction landmark events its history, various platforms, systems, First, discuss history genome-editing. Second, describe current state strategies explain how techniques offer unprecedented precision versatility modifying human genome. Third, introduce systems used deploy components through DNA, RNA, RNPs. Finally, summarize labeling endogenous genes, screening variants, molecular recording, disease models, gene therapy, ex therapy potential future advances.

Language: Английский

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Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations

Nature Genetics, Journal Year: 2025, Volume and Issue: 57(1), P. 140 - 153

Published: Jan. 1, 2025

Abstract The mutational landscape of TP53 , a tumor suppressor mutated in about half all cancers, includes over 2,000 known missense mutations. To fully leverage mutation status for personalized medicine, thorough understanding the functional diversity these mutations is essential. We conducted deep scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 variants cancer cells. This high-resolution approach, covering 94.5% cancer-associated mutations, precisely mapped impact individual on cell fitness, surpassing previous studies distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants pharmacological reactivation. Moreover, we uncovered roles splicing alterations nonsense-mediated messenger RNA decay mutation-driven dysfunction. These findings underscore power advancing clinical variant interpretation genetic counseling therapy.

Language: Английский

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Saturation genome editing-based clinical classification of BRCA2 variants

Nature, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 8, 2025

Language: Английский

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Functional evaluation and clinical classification of BRCA2 variants

Nature, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 8, 2025

Germline BRCA2 loss-of function variants, which can be identified through clinical genetic testing, predispose to several cancers1–5. However, variants of uncertain significance limit the utility test results. Thus, there is a need for functional characterization and classification all facilitate management individuals with these variants. Here we analysed possible single-nucleotide from exons 15 26 that encode DNA-binding domain hotspot pathogenic missense To enable this, used saturation genome editing CRISPR–Cas9-based knock-in endogenous targeting human haploid HAP1 cells6. The assay was calibrated relative nonsense silent validated using benign standards ClinVar results homology-directed repair assay7. Variants (6,959 out 6,960 evaluated) were assigned seven categories pathogenicity based on VarCall Bayesian model8. Single-nucleotide loss-of-function associated increased risks breast cancer ovarian cancer. integrated into models ClinGen, American College Medical Genetics Genomics, Association Molecular Pathology9 Using this approach, 91% classified as or likely benign. These improve variant. Results comprehensive evaluation particularly significance, provide useful resource who carry such

Language: Английский

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Scalable Functional Assays for the Interpretation of Human Genetic Variation

Annual Review of Genetics, Journal Year: 2022, Volume and Issue: 56(1), P. 441 - 465

Published: Sept. 3, 2022

Scalable sequence-function studies have enabled the systematic analysis and cataloging of hundreds thousands coding noncoding genetic variants in human genome. This has improved clinical variant interpretation provided insights into molecular, biophysical, cellular effects at an astonishing scale resolution across spectrum allele frequencies. In this review, we explore current applications prospects for field outline principles underlying scalable functional assay design, with a focus on study single-nucleotide variants.

Language: Английский

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Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2

Nature Communications, Journal Year: 2022, Volume and Issue: 13(1)

Published: March 14, 2022

Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility CRISPR technology. Here, we assess activity specificity WT-Cas9 10 by benchmarking their PAM preferences, on-target activity, off-target susceptibility in cell culture assays with thousands guides targeting endogenous genes. To enhance coverage thus base editing screens, demonstrate that SpCas9-NG SpG are compatible both A > G C T editors, more than tripling number assayable residues. We performance these technologies screening for loss-of-function mutations BRCA1 Venetoclax-resistant BCL2, identifying known new alter function. anticipate tools methodologies described here will facilitate investigation genetic at a finer deeper resolution any locus interest.

Language: Английский

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High-throughput PRIME-editing screens identify functional DNA variants in the human genome

Molecular Cell, Journal Year: 2023, Volume and Issue: 83(24), P. 4633 - 4645.e9

Published: Dec. 1, 2023

Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact a high-throughput and single-base resolution manner remains challenging. Here, we develop pooled prime-editing screen method, PRIME, that can be applied to characterize thousands of coding non-coding single experiment high reproducibility. To showcase its applications, first identified essential nucleotides for 716 bp MYC enhancer via PRIME-mediated analysis. Next, PRIME functionally 1,304 genome-wide association study (GWAS)-identified breast cancer 3,699 from ClinVar. We discovered 103 156 uncertain significance are affecting cell fitness. Collectively, demonstrate is capable characterizing genetic at scale, advancing accurate genome annotation disease risk prediction, diagnosis, therapeutic target identification.

Language: Английский

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A prime editor mouse to model a broad spectrum of somatic mutations in vivo

Nature Biotechnology, Journal Year: 2023, Volume and Issue: unknown

Published: May 11, 2023

Language: Английский

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