Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations

Nature Genetics, Год журнала: 2025, Номер 57(1), С. 140 - 153

Опубликована: Янв. 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.

Язык: Английский

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High-throughput screening of human genetic variants by pooled prime editing

Cell Genomics, Год журнала: 2025, Номер unknown, С. 100814 - 100814

Опубликована: Март 1, 2025

Язык: Английский

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Multimodal scanning of genetic variants with base and prime editing

Nature Biotechnology, Год журнала: 2024, Номер unknown

Опубликована: Ноя. 12, 2024

Mutational scanning connects genetic variants to phenotype, enabling the interrogation of protein functions, interactions and variant pathogenicity. However, current methodologies cannot efficiently engineer customizable sets diverse in endogenous loci across cellular contexts high throughput. Here, we combine cytosine adenine base editors a prime editor assess pathogenicity broad spectrum epithelial growth factor receptor gene (EGFR). Using pooled editing guide RNA libraries, install tens thousands spanning full coding sequence EGFR multiple cell lines role these tumorigenesis resistance tyrosine kinase inhibitors. Our scan identifies important hits, supporting robustness approach revealing underappreciated routes activation drug response. We anticipate that multimodal precision mutational can be applied broadly characterize variation any element interest at single-nucleotide resolution.

Язык: Английский

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Open-ended molecular recording of sequential cellular events into DNA

Nature Chemical Biology, Год журнала: 2024, Номер unknown

Опубликована: Ноя. 14, 2024

Язык: Английский

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A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening

Nature Methods, Год журнала: 2024, Номер unknown

Опубликована: Ноя. 19, 2024

Prime editing installs precise edits into the genome with minimal unwanted byproducts, but low and variable efficiencies have complicated application of approach to high-throughput functional genomics. Here we assembled a prime platform capable high-efficiency substitution suitable for interrogation small genetic variants. We benchmarked this pooled, loss-of-function screening using library ~240,000 engineered guide RNAs (epegRNAs) targeting ~17,000 codons 1–3 bp substitutions. Comparing abundance these epegRNAs across screen samples identified negative selection phenotypes 7,996 nonsense mutations targeted 1,149 essential genes synonymous that disrupted splice site motifs at 3′ exon boundaries. Rigorous evaluation codon-matched controls demonstrated were highly specific intended edit. Altogether, established multiplexed, characterization variants simple readouts. This work establishes (up tens thousands) phenotypes.

Язык: Английский

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Accelerated drug-resistant variant discovery with an enhanced, scalable mutagenic base editor platform

Cell Reports, Год журнала: 2024, Номер 43(6), С. 114313 - 114313

Опубликована: Июнь 1, 2024

Personalized cancer therapeutics bring directed treatment options to patients based on their tumor's genetic signature. Unfortunately, tumor genomes are remarkably adaptable, and acquired resistance through gene mutation frequently occurs. Identifying mutations that promote within drug-treated patient populations can be cost, resource, time intensive. Accordingly, base editing, enabled by Cas9-deaminase domain fusions, has emerged as a promising approach for rapid, large-scale variant screening in situ. Here, we adapt optimize conditional activation-induced cytidine deaminase (AID)-dead Cas9 (dCas9) system, which demonstrates greater heterogeneity of edits with an expanded footprint compared the most commonly utilized cytosine editor, BE4. In combination custom single guide RNA (sgRNA) library, identify individual compound variants epidermal growth factor receptor (EGFR) v-raf murine sarcoma viral oncogene homolog B1 (BRAF) confer established EGFR inhibitors. This system analytical pipeline provide simple, highly scalable platform cis or trans drug-modifying discovery uncovering valuable insights into protein structure-function relationships.

Язык: Английский

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Prime editing sensors enable multiplexed genome editing

Nature Reviews Genetics, Год журнала: 2024, Номер 25(7), С. 454 - 454

Опубликована: Апрель 29, 2024

Язык: Английский

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Analyzing the functional effects of DNA variants with gene editing

Cell Reports Methods, Год журнала: 2024, Номер 4(5), С. 100776 - 100776

Опубликована: Май 1, 2024

Continual advancements in genomics have led to an ever-widening disparity between the rate of discovery genetic variants and our current understanding their functions potential roles disease. Systematic methods for phenotyping DNA are required effectively translate data into improved outcomes patients with diseases. To make biggest impact, these approaches must be scalable accurate, faithfully reflect disease biology, define complex mechanisms. We compare analyze function endogenous context using genome editing strategies, such as saturation editing, base prime editing. discuss how technologies can linked high-content readouts gain deep mechanistic insights variant effects. Finally, we highlight key challenges that need addressed bridge genotype phenotype gap, ultimately improve diagnosis treatment

Язык: Английский

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Computational modeling of human genetic variants in mice

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Фев. 24, 2025

Mouse models represent a powerful platform to study genes and variants associated with human diseases. While genome editing technologies have increased the rate precision of model development, predicting installing specific types mutations in mice that mimic native genetic context is complicated. Computational tools can identify align orthologous wild-type sequences from different species; however, predictive modeling engineering equivalent mouse mirror nucleotide and/or polypeptide change effects remains challenging. Here, we present H2M (human-to-mouse), computational pipeline analyze variation data systematically predict functional consequences variants. We show integrate mouse-to-human paralog-to-paralog variant mapping analyses pipelines devise strategies tailored mice. leveraged these establish database containing > 3 million human-mouse mutation pairs, as well silico -designed base prime libraries engineer 4,944 recurrent pairs. Using H2M, also found predicted pathogenicity immunogenicity scores were highly correlated between suggesting similar sequence may exhibit broad interspecies conservation. Overall, fills gap field by establishing robust versatile framework homologous across species while providing key experimental resources augment genetics medicine applications. The (including software package documentation) be accessed at https://human2mouse.com .

Язык: Английский

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Multiplexed in vivo base editing identifies functional gene-variant-context interactions

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Фев. 26, 2025

Human genome sequencing efforts in healthy and diseased individuals continue to identify a broad spectrum of genetic variants associated with predisposition, progression, therapeutic outcomes for diseases like cancer 1-6 . Insights derived from these studies have significant potential guide clinical diagnoses treatment decisions; however, the relative importance functional impact most remain poorly understood. Precision editing technologies base prime can be used systematically engineer interrogate diverse types endogenous their native context 7-9 We others recently developed applied scalable sensor-based screening approaches measure phenotypes produced by thousands mutations vitro 10-12 However, physiological vivo setting, including contextual differences depending on tissue or microenvironment, remains unexplored. Here, we integrate new cross-species sensor libraries syngeneic mouse models develop multiplexed platform systematic analysis primary disseminated malignancies. this screen 13,840 RNAs designed 7,783 human cancer-associated mapping 489 protein-coding genes, allowing us construct rich compendium putative interactions between mutations, contexts. Our findings suggest that environment cellular organotropism are important determinants specific gene-variant phenotypes. also show many effects fail detected standard CRISPR-Cas9 nuclease often produce discordant phenotypes, potentially due site-specific amino acid selection- separation-of-function mechanisms. This versatile could deployed investigate how variation impacts other diseases, as well avenues treat disease.

Язык: Английский

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