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

Cell Genomics, Journal Year: 2025, Volume and Issue: unknown, P. 100814 - 100814

Published: March 1, 2025

Language: Английский

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Rewriting regulatory DNA to dissect and reprogram gene expression

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 21, 2023

Regulatory DNA sequences within enhancers and promoters bind transcription factors to encode cell type-specific patterns of gene expression. However, the regulatory effects programmability such remain difficult map or predict because we have lacked scalable methods precisely edit quantify in an endogenous genomic context. Here present approach measure quantitative hundreds designed sequence variants on expression, by combining pooled CRISPR prime editing with RNA fluorescence

Language: Английский

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

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

Published: Nov. 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.

Language: Английский

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Analyzing the functional effects of DNA variants with gene editing

Cell Reports Methods, Journal Year: 2024, Volume and Issue: 4(5), P. 100776 - 100776

Published: May 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

Language: Английский

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Rewriting regulatory DNA to dissect and reprogram gene expression

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

Published: April 1, 2025

Language: Английский

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Deconstructing cancer with precision genome editing

Biochemical Society Transactions, Journal Year: 2024, Volume and Issue: 52(2), P. 803 - 819

Published: April 17, 2024

Recent advances in genome editing technologies are allowing investigators to engineer and study cancer-associated mutations their endogenous genetic contexts with high precision efficiency. Of these, base prime quickly becoming gold-standards the field due versatility scalability. Here, we review merits limitations of these technologies, application modern cancer research, speculate how could be integrated address future directions field.

Language: Английский

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Saturation profiling of drug-resistant genetic variants using prime editing

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

Published: Nov. 12, 2024

Language: Английский

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

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 27, 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. Leveraging several recent advances, we assembled a prime platform capable high-efficiency substitution across set engineered guide RNAs (epegRNAs) corresponding target sequences (80% median intended editing). Then, using custom library 240,000 epegRNAs targeting >17,000 codons 175 different types, benchmarked our for interrogation small variants (1-3 nucleotides) targeted essential genes. Resulting data identified negative growth phenotypes nonsense mutations ~8,000 codons, comparing those results from controls demonstrated high specificity. We also observed synonymous that disrupted splice site motifs at 3' exon boundaries. Altogether, establish benchmark characterization genetic simple readouts multiplexed experiments.

Language: Английский

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High throughput evaluation of genetic variants with prime editing sensor libraries

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2022, Volume and Issue: unknown

Published: Oct. 26, 2022

Abstract Many human diseases have a strong association with diverse types of genetic alterations. These include cancer, in which tumor genomes often harbor complex spectrum single-nucleotide alterations and chromosomal rearrangements that can perturb gene function ways remain poorly understood. Some cancer-associated genes exhibit tremendous degree mutational heterogeneity, may impact disease initiation, progression, therapy responses. For example, TP53 , the most frequently mutated shows extensive allelic variation leads to generation altered proteins produce functionally distinct phenotypes. Whether variants other encode loss-of-function, gain-of-function, or otherwise neomorphic phenotypes remains both controversial technically challenging assess, particularly at endogenous level. Here, we present high-throughput prime editing “sensor” strategy quantitatively assess functional variants. We used this screen largest collection assembled date, identifying known novel alleles p53 mechanistically ways. Intriguingly, find certain variants, those oligomerization domain, display opposite exogenous overexpression systems. disease-relevant found humans cancer predisposition syndromes unique molecular properties. Our results emphasize physiological importance dosage shaping native protein stoichiometry protein-protein interactions, highlight dangers using systems interpret pathogenic alleles, establish powerful computational experimental framework for studying their sequence context scale.

Language: Английский

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