High throughput CRISPR approaches: New solutions for challenging problems

TrAC Trends in Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown, P. 118245 - 118245

Published: March 1, 2025

Language: Английский

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Multimodal scanning of genetic variants with base and prime editing

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

Published: Nov. 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.

Language: Английский

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Aggressive Serous Carcinomas of the Female Reproductive Tract: Cancer-Prone Cell States and Genetic Drivers

Cancers, Journal Year: 2025, Volume and Issue: 17(4), P. 604 - 604

Published: Feb. 11, 2025

In 2025, gynecological cancers are projected to account for approximately 10% of cancer-related deaths in women. High-grade serous ovarian carcinoma (HGSC) and endometrial (SEC) the most lethal cancer subtypes. Both malignancies commonly have TP53 mutations, alterations RB1 pathway, numerous secondary mutations. types consist poorly differentiated highly heterogeneous cell populations at time detection. Latent development rapid progression HGSC SEC impede identification definitive cells origin genetic drivers. Here, we review our current knowledge about cancer-prone states We also discuss how emerging transcriptomic tools applied contemporary model systems may facilitate novel targets timely detection therapeutic intervention.

Language: Английский

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

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 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.

Language: Английский

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Using Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries

Methods in enzymology on CD-ROM/Methods in enzymology, Journal Year: 2025, Volume and Issue: unknown, P. 437 - 451

Published: Jan. 1, 2025

Language: Английский

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CRISPR/Cas technologies for cancer drug discovery and treatment

Trends in Pharmacological Sciences, Journal Year: 2025, Volume and Issue: unknown

Published: March 1, 2025

Language: Английский

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Mechanistic Insights into Proteomic Mutation-Phenotype Linkages from Tiling Mutagenesis Screens

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: April 23, 2025

ABSTRACT High-throughput mutagenesis screens are powerful tools for mapping mutations to phenotypes. However, deciphering the molecular mechanisms that link phenotypic outcomes remains a significant challenge. Here, we present ProTiler-Mut, versatile computational framework harnesses tiling screens, which introduce variants across entire protein sequences, facilitate investigation of mutation-to-phenotype associations at multiple levels, including individual residues, substructures, and protein-protein interactions (PPIs). As demonstrated through our analyses base editing (BE) targeting DNA Damage Response (DDR) proteins T cell regulators, ProTiler-Mut provides novel insights into mutation-phenotype linkages, including: i) refined classification mutation reveals separation-of-function (SOF) category beyond conventional binary loss-of-function (LOF) gain-of-function (GOF); ii) definition phenotype-associated hotspot substructures enable inference function unscreened pathogenic mutations; iii) identification PPIs disrupted by functional mutations. Through analyses, identified substructure harboring GOF disrupt between kinases MAPK1 RSK1, leading activation elevated expression immune checkpoint receptor PD-1. Furthermore, demonstrate applicability various screening platforms, highlighting its broad utility generalizability.

Language: Английский

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BEscreen: a versatile toolkit to design base editing libraries

Nucleic Acids Research, Journal Year: 2025, Volume and Issue: unknown

Published: May 3, 2025

Base editing enables the high-throughput screening of genetic variants for phenotypic effects. screens require design single guide RNA (sgRNA) libraries to enable either gene- or variant-centric approaches. While computational tools supporting sgRNAs exist, no solution offers versatile and scalable library enabling all major use cases. Here, we introduce BEscreen, a comprehensive base tool provided as web server (bescreen.ostendorflab.org) command line tool. BEscreen provides variant-, gene-, region-centric modes accommodate various The variant mode accepts genomic coordinates, amino acid changes, rsIDs input. gene designs near-saturation covering entire coding sequence given genes transcripts, region possible guides regions. selection by biological consequence, it features customization editor characteristics, optional annotation using Ensembl's Variant Effect Predictor. In sum, is highly wide range cases with seamless scalability from individual large, libraries.

Language: Английский

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Precision mutational scanning: your multipass to the future of genetics

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

Published: Nov. 19, 2024

Language: Английский

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