Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells DOI Open Access
Júlia Daher Carneiro Marsiglia,

Kia Vaalavirta,

Estefany Knight

et al.

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

Published: Dec. 13, 2023

Abstract Promiscuous editing by CRISPR/Cas systems within the human genome is a major challenge that must be addressed prior to applying these therapeutically. In bacteria, have evolved in co-evolutionary arms race with infectious phage viruses contain inhibitory anti-CRISPR proteins their genomes. Here, we harness outcome of this engineer an AcrIIA4 protein increase precision CRISPR/Cas-based targeting. We developed approach specifically leveraged (1) language models, (2) deep mutational scanning, and (3) highly parallel DNA repair measurements cells. single experiment, ∼10,000 variants were tested identify lead eliminated detectable off-target events while retaining on-target activity. The candidates further focused round screening included high-fidelity version Cas9 as benchmark. Finally, arrayed experiments using delivered ribonucleoprotein conducted demonstrated gene across two independent genomic loci reduction frequency translocation between site. Thus, language-model-guided high-throughput effective way efficiently precision, which could used improve fidelity editing-based therapeutics reduce genotoxicity.

Language: Английский

Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations DOI Creative Commons
Julianne Funk,

Maria Klimovich,

Daniel Drangenstein

et al.

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

Citations

8
dsDAP: An efficient method for high-abundance DNA-encoded library construction in mammalian cells DOI
Kaili Zhang, Yiping Wang,

S. S. Jiang

et al.

International Journal of Biological Macromolecules, Journal Year: 2025, Volume and Issue: 298, P. 140089 - 140089

Published: Jan. 20, 2025

Language: Английский

Citations

0
Redefining antibody patent protection using paratope mapping and CDR-scanning DOI

Soma S. R. Banik,

Xiaoxiang Deng, Edgar Davidson

et al.

Nature Biotechnology, Journal Year: 2025, Volume and Issue: 43(2), P. 170 - 174

Published: Feb. 1, 2025

Language: Английский

Citations

0
Biased Signaling in G Protein-Coupled Receptors: Understanding the Biological Relevance and Tools for Probing Functionally Selective Ligands DOI
Braden S. Fallon,

Kathleen E. Rondem,

Elizabeth J. Mumby

et al.

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

Published: March 18, 2025

Biased signaling has transformed pharmacology by revealing that receptors, particularly G protein-coupled receptors (GPCRs), can activate specific intracellular pathways selectively rather than uniformly. This discovery enables the development of targeted therapeutics minimize side effects precisely modulating receptor activity. Functionally selective ligands, which preferentially distinct branches, have become essential tools for exploring mechanisms and uncovering complexities GPCR signaling. These ligands help clarify function in various physiological pathological contexts, offering profound implications therapeutic innovation. GPCRs, mediate a wide range cellular responses through coupling to proteins arrestins, are key pharmacological targets, with nearly third FDA-approved drugs acting on them. Recent advancements biosensor development, multiplex assay platforms, deep mutational scanning methods improving our ability define signaling, allowing better understanding biased pathways.

Language: Английский

Citations

0
Mutational scanning ofCRXclassifies clinical variants and reveals biochemical properties of the transcriptional effector domain DOI Creative Commons
James L. Shepherdson,

David M. Granas,

Jie Li

et al.

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

Published: March 27, 2024

Abstract Cone-Rod Homeobox, encoded by CRX , is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Structurally, comprises an ordered DNA-binding homeodomain intrinsically disordered transcriptional effector domain. Although handful human variants in have been shown to cause several different degenerative retinopathies with varying cone rod predominance, as most disease genes vast majority observed genetic are uncharacterized uncertain significance (VUS). We performed deep mutational scan (DMS) nearly all possible single amino acid substitution CRX, using engineered cell-based reporter assay. measured ability each missense variant transactivate synthetic fluorescent construct pooled fluorescence-activated cell sorting assay compared activation strength that wild-type compute activity score, identifying thousands altered activity. calculated statistical confidence score derived from multiple independent measurements marked unique sequence barcodes, curating high-confidence list 2,000 significantly CRX. evaluated performance DMS clinical classification tool gold-standard classified ClinVar, determined scores could be used identify pathogenic high specificity. That this achieved foreign type, even highly type-specific TF like suggests approach shows promise other TFs function types not easily accessible. Per-position average closely aligned predicted structure demonstrated position-specific residue requirements. The domain, contrast, displayed qualitatively pattern effects, following compositional constraints without specific position requirements peptide chain. domain were consistent acidic exposure model activation. Together, results molecular features demonstrate utility classification.

Language: Английский

Citations

2
Computationally guided high-throughput engineering of an anti-CRISPR protein for precise genome editing in human cells DOI Creative Commons
Júlia Daher Carneiro Marsiglia,

Kia Vaalavirta,

Estefany Knight

et al.

Cell Reports Methods, Journal Year: 2024, Volume and Issue: 4(10), P. 100882 - 100882

Published: Oct. 1, 2024

Language: Английский

Citations

2
Epitope mapping via in vitro deep mutational scanning methods and its applications DOI Creative Commons

Meredith M. Keen,

A. Keith, Eric A. Ortlund

et al.

Journal of Biological Chemistry, Journal Year: 2024, Volume and Issue: unknown, P. 108072 - 108072

Published: Dec. 1, 2024

Language: Английский

Citations

2
Venous Thromboembolic Disease Genetics: From variants to function DOI Open Access
Mary Underwood,

Clint R. Bidlack,

Karl C. Desch

et al.

Journal of Thrombosis and Haemostasis, Journal Year: 2024, Volume and Issue: 22(9), P. 2393 - 2403

Published: Sept. 1, 2024

Language: Английский

Citations

1
In silico and biological analyses of missense variants of the human biliary efflux transporter ABCC2: effects of novel rare missense variants DOI Open Access
Charlotte Kölz, Fabienne Z. Gaugaz, Niklas Handin

et al.

British Journal of Pharmacology, Journal Year: 2024, Volume and Issue: 181(22), P. 4593 - 4609

Published: Aug. 2, 2024

Abstract Background and Purpose The ATP‐dependent biliary efflux transporter ABCC2, also known as multidrug resistance protein 2 (MRP2), is essential for the cellular disposition detoxification of various xenobiotics including drugs well endogenous metabolites. Common functionally relevant ABCC2 genetic variants significantly alter drug responses contribute to side effects. aim this study was determine functional consequences rare identified in subjects with European ancestry using silico tools vitro analyses. Experimental Approach Targeted next‐generation sequencing gene used identify novel (n = 143). Twenty‐six were predict consequences. For biological validation, transport assays carried out membrane vesicles prepared from cell lines overexpressing newly estradiol β‐glucuronide carboxydichlorofluorescein substrates. Key Results Three missense (W227R, K402T, V489F). Twenty‐five predicted W227R damaging one potentially damaging. Prediction not possible K402T V489F common linked V1188E/C1515Y. Characterisation showed increased function W227R, V1188E/C1515Y both substrates, whereas only carboxydichlorofluorescein. Conclusion Implications In unable accurately substrate‐dependent increase variants. studies are required activity avoid misleading therapy.

Language: Английский

Citations

0
Navigating the Fitness Landscapes of Plasmodium falciparum Dihydrofolate Reductase: Evolutionary Insights into Antifolate Resistance DOI Open Access

Danny Muzata,

Dwipanjan Sanyal,

Arti Shivram

et al.

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

Published: Nov. 3, 2024

Abstract The rapid emergence of drug resistance in malaria parasites poses a significant challenge to the efficacy antifolate treatments. Traditional development approaches, which often rely on empirical screening with limited mechanistic insights, tend overlook complex evolutionary mechanisms that enable Plasmodium falciparum evade inhibition while preserving enzyme functionality. In this study, we employed computational techniques investigate mutational landscape dihydrofolate reductase (DHFR), focusing regions essential for stability and resistance. Our analysis uncovered conserved residues stability, mutation hotspots enhance adaptability under pressure co-evolving clusters revealing critical functional interdependencies. Through integrated approaches including scanning, epistatic interaction modeling, fitness trajectory mapping, elucidated distinct pathways drive We were able track adaptive paths taken by wild-type upon mutation, steps required reach high-fitness peaks within rugged landscape. These findings provide valuable insights into molecular suggest future design should target networks support next-generation therapies overcome

Language: Английский

Citations

0