Deep mutational scanning of proteins in mammalian cells

Maes, Stefanie; Deploey, Nick; Peelman, Frank; Eyckerman, Sven

doi:10.1016/j.crmeth.2023.100641

Cited by 3 publications

(1 citation statement)

References 166 publications

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“…The approach used in this study, with a synthetic transcriptional reporter and non-native cell type, may be broadly applicable to other TFs with expression limited to specific cell types that are difficult to obtain or culture. Compared to the growth and survival assays that are more typical of deep mutational scans in mammalian cells (Maes et al 2023), transcriptional reporters have the benefit of being a direct readout of TF protein activity. This may help to increase their sensitivity to the effects of protein variants, even in a non-physiologic context.…”

Section: Discussionmentioning

confidence: 99%

Mutational scanning ofCRXclassifies clinical variants and reveals biochemical properties of the transcriptional effector domain

Shepherdson,

Granas,

et al. 2024

Preprint

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Cone-Rod Homeobox, encoded by CRX, is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Structurally, CRX comprises an ordered DNA-binding homeodomain and an intrinsically disordered transcriptional effector domain. Although a handful of human variants in CRX have been shown to cause several different degenerative retinopathies with varying cone and rod predominance, as with most human disease genes the vast majority of observed CRX genetic variants are uncharacterized variants of uncertain significance (VUS). We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitution variants in CRX, using an engineered cell-based transcriptional reporter assay. We measured the ability of each CRX missense variant to transactivate a synthetic fluorescent reporter construct in a pooled fluorescence-activated cell sorting assay and compared the activation strength of each variant to that of wild-type CRX to compute an activity score, identifying thousands of variants with altered transcriptional activity. We calculated a statistical confidence for each activity score derived from multiple independent measurements of each variant marked by unique sequence barcodes, curating a high-confidence list of over 1,800 variants with significantly altered transcriptional activity compared to wild-type CRX. We evaluated the performance of the DMS assay as a clinical variant classification tool using gold-standard classified human variants from ClinVar, and determined that activity scores could be used to identify pathogenic variants with high specificity. That this performance could be achieved using a synthetic reporter assay in a foreign cell type, even for a highly cell type-specific TF like CRX, suggests that this approach shows promise for DMS of other TFs that function in cell types that are not easily accessible. Per-position average activity scores closely aligned to a predicted structure of the ordered homeodomain and demonstrated position-specific residue requirements. The intrinsically disordered transcriptional effector domain, by contrast, displayed a qualitatively different pattern of substitution effects, following compositional constraints without specific residue position requirements in the peptide chain. The observed compositional constraints of the effector domain were consistent with the acidic exposure model of transcriptional activation. Together, the results of the CRX DMS identify molecular features of the CRX effector domain and demonstrate clinical utility for variant classification.

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Section: Discussionmentioning

confidence: 99%

Mutational scanning ofCRXclassifies clinical variants and reveals biochemical properties of the transcriptional effector domain

Shepherdson,

Granas,

et al. 2024

Preprint

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Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

Marsiglia,

Vaalavirta,

Knight

et al. 2023

Preprint

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

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In silico and biological analyses of missense variants of the human biliary efflux transporter ABCC2: effects of novel rare missense variants

Kölz,

Gaugaz,

Handin

et al. 2024

British J Pharmacology

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

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Deep mutational scanning of proteins in mammalian cells

Cited by 3 publications

References 166 publications

Mutational scanning ofCRXclassifies clinical variants and reveals biochemical properties of the transcriptional effector domain

Mutational scanning ofCRXclassifies clinical variants and reveals biochemical properties of the transcriptional effector domain

Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

In silico and biological analyses of missense variants of the human biliary efflux transporter ABCC2: effects of novel rare missense variants

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