Biology subjects

Starita, L. M.

Publications and source records attributed to Starita, L. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Massively scaled variant effect mapping across the MAPK pathway reveals the importance of molecular and cellular context

Variation alters inherent protein properties like thermodynamic stability and catalytic activity, and these intrinsic effects are shaped by the complex cellular environment. Signaling cascades typify this complexity, with tens or hundreds of proteins acting in concert through regulated states and interactions to transduce signals. Here, we measure the effect of 186,676 variants across 17 proteins in the RAS-MAPK pathway on protein abundance and pathway activity in human cells under diverse conditions, for a total of 503,099 variant effect measurements. We discovered 14,412 dominant negative variants across 12 proteins, nearly all previously unknown, that disrupt specific domains or interaction interfaces while leaving other functions intact and that are depleted from the human population. Measuring the abundance of 106,642 variants across 10 proteins under HSP90 inhibition showed that HSP90 shapes the effect of 67% of missense variants across the pathway, buffering destabilizing variants in both strong and weak clients. Our variant effect data, collected in the context of an intact proteome, enabled us to assess the functional importance of 395 experimentally and computationally modeled MAPK protein interactions. Using additional mutational scans, we resolved how the promiscuous switch II helix binding surface of KRAS accommodates overlapping interactions with LZTR1 and SOS1, revealing distinct allosteric networks that support each interaction. Thus, we establish a framework for measuring variant effects at unprecedented scale in human cells, illuminating fundamental MAPK pathway signaling mechanisms and demonstrating that cellular context profoundly shapes the effect of most variants.

genomics↗

bioRxiv2026

Gene- and domain-aware calibration increases the clinical utility of variant effect predictors

The utility of clinical genetic testing is limited because around 90% of missense variants in ClinVar remain of uncertain clinical significance. Variant effect predictors (VEPs) can score any missense variant, potentially empowering variant classification. Realizing this potential requires calibration to translate predictor scores into evidence. However, genome-wide calibration ignores predictor heterogeneity across genes, causing evidence misassignment. We developed an automated, data-adaptive framework that optimizes two complementary approaches: gene-specific calibration for genes with enough variants for calibration, and domain-aggregate calibration for other disease-associated genes, which groups variants from protein domains with similar predictor score distributions for calibration. Applied to three predictors across 2,769 genes, this framework assigned evidence to 10.6% more variants on average while generally improving evidence accuracy compared to genome-wide calibration. These calibrations and the resulting calibrated computational evidence are available through the PredictMD portal. Our framework substantially increases the clinical utility of VEPs for variant classification.

genetics↗

bioRxiv2026

An integrated, scaled approach to resolve TSC2 variants of uncertain significance

Obtaining a precise genetic tuberous sclerosis diagnosis is a challenge as many missense TSC2 variants are variants of uncertain significance (VUS). VUS in TSC2 have been resolved by one-at-a-time functional assays, but these assays cannot scale to the 3,634 TSC2 missense VUS observed so far. To address this challenge, we used massively parallel sequencing to measure the steady-state abundance of almost 9,000 TSC2 missense variants and developed an mTOR pathway activity assay using genome editing and cell sorting to generate activity scores for 391 missense variants. 1,288 of 8,891 (14.49%) missense variants assayed had altered TSC2 abundance, and 69 of 391 (17.65%) missense variants assayed had altered mTOR pathway activity. Calibration and integration of these data into classification of variants identified in a clinical cohort putatively reclassified 212 of 276 (76.8%) TSC2 missense VUS. These datasets will lead to improved genetic diagnosis of tuberous sclerosis with potential positive impacts on the clinical management of patients and their families.

genetics↗

bioRxiv2026

High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

The tumor suppressor RAD51D is essential for homologous recombination (HR). Pathogenic variants in RAD51D are associated with breast and ovarian cancers. However, most clinical missense variants are of unknown significance. We performed a multiplex assay of variant effect to test 6,888 RAD51D coding variants for loss-of-function. The resulting variant-to-function map perfectly separates known pathogenic and benign variants and is validated by orthogonal HR and biochemical assays across 70 clinical variants. Our screen shows that variants in the DNA-binding or ATPase core most severely compromise HR, and we identify the RAD51D-RAD51C interface within the BCDX2 complex as essential for regulating its ATPase activity. We hypothesize that, paradoxically, the primary function of RAD51D is to slow the ATPase activity of BCDX2, thereby allowing sufficient time and space for RAD51 filament assembly. Together, we identify hotspots of deleterious RAD51D variants and uncover the mechanisms by which variants compromise its biochemical functions. HighlightsO_LIUsed a multiplexed assay of functional effect (MAVE) to assess the functionality via olaparib sensitivity of 6,888 RAD51D coding variants, which can be used for variant classification C_LIO_LIProvided cellular functional analysis for 70 clinically-identified breast and ovarian cancer RAD51D variants C_LIO_LIIdentified key regions and enzymatic activities of RAD51D critical for its function in the BCDX2 and the X3CDX2 complexes C_LIO_LIDetermined mechanism of RAD51D-mediated regulation of BCDX2 ATPase activity C_LI

cancer biology↗

bioRxiv2025

Single-cell, multi-region profiling of the macaque brain across the lifespan

Brain aging is a complex process with profound health and societal consequences. However, the molecular and cellular pathways that govern its temporal progression-along with any cell type-, region-, and sex-specific heterogeneity in such progression-remain poorly defined. Here, we present a transcriptomic atlas of 5.3 million cells from 582 samples spanning 11 brain regions of 55 rhesus macaques (29 female, 26 male), aged 5 months (early life) to 21 years (late adulthood). We annotate 12 major cell classes and 225 subclusters, including region-specific subtypes of excitatory and inhibitory neurons, astrocytes, and ependymal cells. We identify a vulnerable excitatory neuron population in the superficial cortical lamina and a cortical interneuron population that are less abundant later in life, along with subtle, region-specific, age-associated compositional differences in subpopulations of microglia and oligodendrocytes, whose detection required single-cell resolution. Finally, we chart convergent and divergent age-associated molecular signatures across brain regions and cell classes-where some of these signatures are sex-specific and could underlie sex biases in neurological disorders. We find that age-associated transcriptional programs not only overlap substantially with those seen in Alzheimers disease (AD), but also unfold along distinct temporal trajectories across brain regions, suggesting that aging and AD may share molecular roots that emerge at different life stages and in region-specific, sex-specific windows of vulnerability. This work provides a temporal, regional, and sex-stratified atlas of the aging primate brain, offering insights into cell type-specific vulnerabilities and regional heterogeneity with translational human relevance.

neuroscience↗

bioRxiv2025

Image-based, pooled phenotyping reveals multidimensional, disease-specific variant effects

Genetic variants often produce complex phenotypic effects that confound current assays and predictive models. We developed Variant in situ sequencing (VIS-seq), a pooled, image-based method that measures variant effects on molecular and cellular phenotypes in diverse cell types. Applying VIS-seq to [~]3,000 LMNA and PTEN variants yielded high-dimensional morphological profiles that captured variant-driven changes in protein abundance, localization, activity and cell architecture. We identified gain-of-function LMNA variants that reshape the nucleus and autism-associated PTEN variants that mislocalize. Morphological profiles predicted variant pathogenicity with near-perfect accuracy and distinguished autism-linked from tumor syndrome-linked PTEN variants. Most variants impacted a multidimensional continuum of phenotypes not recapitulated by any single functional readout. By linking protein variation to cell images at scale, we illuminate how variant effects cascade from molecular to subcellular to cell morphological phenotypes, providing a framework for resolving the complexity of variant function.

genomics↗

bioRxiv2025

Principles and functional consequences of plasmid chromatinization in mammalian cells

Plasmids have fundamentally transformed how we resolve regulatory grammar across the tree of life. However, although chromatin plays an integral role in regulating the function of regulatory elements along the nuclear genome, our understanding of how, or whether, similar chromatin structures form on plasmids transfected into mammalian cells remains limited. We demonstrate that plasmid single-molecule chromatin fiber sequencing (plasmid Fiber-seq) can accurately map chromatin architectures along individual, full-length transfected plasmid molecules at near single-nucleotide resolution. Application of plasmid Fiber-seq to diverse plasmids and cell lines demonstrates that plasmids are chromatinized in a sequence-dependent organized manner and adopt a heterogeneous and incomplete chromatin architecture relative to nuclear-encoded chromatin fibers. We show that the focal occupancy of nucleosomes and transcription factors along transfected plasmids is central to their transcriptional activity within mammalian cells, and demonstrate that plasmids indeed are capable of recapitulating nuclear genome-encoded chromatin architectures, although not always. Finally, we demonstrate that combining plasmid Fiber-seq with high-throughput reporter assays can establish the molecular mechanisms underlying pathogenic non-coding variants, including disentangling the effects of transcriptional activators and repressors with near-single-nucleotide resolution. Overall, our findings reveal the principles by which plasmid-based assays can be used for accurate fine-scale mapping of chromatin-dependent regulatory grammar.

genomics↗

bioRxiv2025

Gene-based calibration of high-throughput functional assays for clinical variant classification

High-throughput assays measure a broad range of variant effects on gene function and hold promise for supporting genomic medicine. Current clinical guidelines for rare Mendelian diseases rely on establishing gene-specific score thresholds for each assay that separate pathogenic from benign variants. This introduces inconsistencies and subjectivity, ultimately lacking the rigor of calibration; i.e., mapping a variant score to a probability of pathogenicity. To address this problem, we introduce a semi-supervised framework for calibrating experimental assay data and propose Experimental score CALIBRator (ExCALIBR), a method that jointly models pathogenic, benign, synonymous, and population variants using skew normal mixtures to produce variant-specific probabilities of pathogenicity. Evaluated across 80 datasets from 39 genes, all meeting fit quality criteria, ExCALIBR substantially outperformed existing field standards and was further validated on the All of Us biobank data. Our results demonstrate that calibrated experimental assays generate indispensable evidence that will dramatically reduce variants of uncertain significance.

bioinformatics↗

bioRxiv2024

The proteomic landscape and temporal dynamics of mammalian gastruloid development

Gastrulation is the process by which the early embryo establishes a body plan and primes itself for organogenesis. As gastrulation is challenging to study in vivo, stem cell-derived "gastruloids" have emerged as powerful surrogates. Although transcriptomics and imaging have been applied extensively to such embryo models, the dynamics of their proteomes remains largely unknown. Here, we apply quantitative proteomics to human and mouse gastruloids at four key stages. We leverage these data to map the expression dynamics of protein complexes, and to nominate cooperative proteins. With matched transcriptome and phosphosite data, we investigate global and stage/pathway-specific discordance between the transcriptome and proteome and nominate kinase-substrate relationships based on phosphosite dynamics. Finally, we apply co-regulation network analysis to identify genes linked to the Commander complex whose perturbation leads to morphological defects in gastruloids. Altogether, our work showcases the potential of applying proteomics to embryo models to advance our understanding of mammalian development in ways challenging through transcriptomics alone.

systems biology↗

bioRxiv2023

Assigning credit where it's due: An information content score to capture the clinical value of Multiplexed Assays of Variant Effect

BackgroundA variant can be pathogenic or benign with relation to a human disease. Current classification categories from benign to pathogenic reflect a probabilistic summary of current understanding. A primary metric of clinical utility for multiplexed assays of variant effect (MAVE) is the number of variants that can be reclassified from uncertain significance (VUS). However, we hypothesized that this measure of utility underrepresents the information gained from MAVEs and that an information theory approach which includes data that does not reclassify variants will better reflect true information gain. We used this information theory approach to evaluate the information gain, in bits, for MAVEs of BRCA1, PTEN, and TP53. Here, one bit represents the amount of information required to completely classify a single variant starting from no information. ResultsBRCA1 MAVEs produced a total of 831.2 bits of information, 6.58% of the total missense information in BRCA1 and a 22-fold increase over the information that only contributed to VUS reclassification. PTEN MAVEs produced 2059.6 bits of information which represents 32.8% of the total missense information in PTEN and an 85-fold increase over the information that contributed to VUS reclassification. TP53 MAVEs produced 277.8 bits of information which represents 6.22% of the total missense information in TP53 and a 3.5-fold increase over the information that contributed to VUS reclassification. ConclusionsAn information content approach will more accurately portray information gained through MAVE mapping efforts than counting the number of variants reclassified. This information content approach may also help define the impact of modifying information definitions used to classify many variants, such as guideline rule changes.

genetics↗

bioRxiv2023

A multiplex, prime editing framework for identifying drug resistance variants at scale

CRISPR-based genome editing has revolutionized functional genomics, enabling screens in which thousands of perturbations of either gene expression or primary genome sequence can be competitively assayed in single experiments. However, for libraries of specific mutations, a challenge of CRISPR-based screening methods such as saturation genome editing is that only one region (e.g. one exon) can be studied per experiment. Here we describe prime-SGE ("prime saturation genome editing"), a new framework based on prime editing, in which libraries of specific mutations can be installed into genes throughout the genome and functionally assessed in a single, multiplex experiment. Prime-SGE is based on quantifying the abundance of prime editing guide RNAs (pegRNAs) in the context of a functional selection, rather than quantifying the mutations themselves. We apply prime-SGE to assay thousands of single nucleotide changes in eight oncogenes for their ability to confer drug resistance to three EGFR tyrosine kinase inhibitors. Although currently restricted to positive selection screens by the limited efficiency of prime editing, our strategy opens the door to the possibility of functionally assaying vast numbers of precise mutations at locations throughout the genome.

genomics↗

bioRxiv2023

Multiplexed functional assessments of MYH7 variants in human cardiomyocytes at scale

BackgroundSingle, autosomal-dominant missense mutations in MYH7, which encodes a sarcomeric protein (MHC-{beta}) in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically-actionable. However, [~]75% of MYH7 variants are of unknown significance (VUS), causing diagnostic challenges for clinicians and emotional distress for patients. Deep mutational scans (DMS) can determine variant effect at scale, but have only been utilized in easily-editable cell lines. While human induced pluripotent stem cells (hiPSCs) can be differentiated to numerous cell types that enable the interrogation of variant effect in a disease-relevant context, DMS have not been executed using diploid hiPSC derivates. However, CRaTER enrichment has recently enabled the pooled generation of a saturated five position MYH7 variant hiPSC library suitable for DMS for the first time. ResultsAs a proof-of-concept, we differentiated this MYH7 variant hiPSC library to cardiomyocytes (hiPSC-CMs) for multiplexed assessment of MHC-{beta} variant abundance by massively parallel sequencing (VAMP-seq) and hiPSC-CM survival. We confirm MHC-{beta} protein loss occurs in a failing human heart with a pathogenic MYH7 mutation. We find the multiplexed assessment of MHC-{beta} abundance and hiPSC-CM survival both accurately segregate all pathogenic variants from synonymous controls. Overall, functional scores of 68 amino acid substitutions across these independent assays are [~]50% consistent. ConclusionsThis study leverages hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of MYH7 missense variants at scale for the first time. This proof-of-concept demonstrates the ability to DMS previously restricted, clinically-actionable genes to reduce the burden of VUS on patients and clinicians.

genetics↗

bioRxiv2023

DNA Repair Function Scores for 2172 Variants in the BRCA1 Amino-Terminus

Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2-192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193-302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2-302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1. AUTHOR SUMMARYMost missense substitutions in BRCA1 are variants of unknown significance (VUS), and individuals with a VUS in BRCA1 cannot know from genetic information alone whether this variant predisposes to breast or ovarian cancer. We apply a multiplexed functional assay for homology directed repair of DNA double strand breaks to assess variant impact on this important BRCA1 protein function. We analyzed 2172 variants in the amino-terminus of BRCA1 and demonstrate that variants that are known as pathogenic have a loss of function in the DNA repair assay. Conversely, variants that are known to be benign are functionally normal in the multiplexed assay. We suggest that these functional determinations of BRCA1 variants can be used to augment the information that clinical cancer geneticists provide to patients who have a VUS in BRCA1.

cancer biology↗

bioRxiv2023

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

CRISPR-based gene activation (CRISPRa) is a promising therapeutic approach for gene therapy, upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate therapeutically relevant genes in a cell type-specific manner.

genomics↗

bioRxiv2023

CRaTER enrichment for on-target gene-editing enables generation of variant libraries in hiPSCs

Standard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describe CRISPRa On-Target Editing Retrieval (CRaTER) which enriches for cells with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of single nucleotide variants (SNVs) in MYH7, a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different MYH7 SNVs. We differentiated these hiPSCs to cardiomyocytes and show MYH7 fusion proteins can localize as expected. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of transgenic cell lines at unprecedented scale.

genetics↗

bioRxiv2022

A single-cell multi-omic atlas spanning the adult rhesus macaque brain

Cataloging the diverse cellular architecture of the primate brain is crucial for understanding cognition, behavior and disease in humans. Here, we generated a brain-wide single-cell multimodal molecular atlas of the rhesus macaque brain. Altogether, we profiled 2.58M transcriptomes and 1.59M epigenomes from single nuclei sampled from 30 regions across the adult brain. Cell composition differed extensively across the brain, revealing cellular signatures of region-specific functions. We also identified 1.19M candidate regulatory elements, many novel, allowing us to explore the landscape of cis-regulatory grammar and neurological disease risk in a cell-type-specific manner. Together, this multi-omic atlas provides an open resource for investigating the evolution of the human brain and identifying novel targets for disease interventions.

neuroscience↗

bioRxiv2022

Single-cell analysis of chromatin and expression reveals age- and sex-associated alterations in the human heart

Sex differences and age-related changes in the human heart at the tissue, cell, and molecular level have been well-documented and many may be relevant for cardiovascular disease. However, how molecular programs within individual cell types vary across individuals by age and sex remains poorly characterized. To better understand this variation, we performed single-nucleus combinatorial indexing (sci) ATAC- and RNA-Seq in human heart samples from nine donors. We identify hundreds of differentially expressed genes by age and sex. Sex dependent alterations include pathways such as TGF{beta} signaling and metabolic shifts by sex, evident in both transcriptional alterations and differing presence of transcription factor (TF) motifs in accessible chromatin. Age was associated with changes such as immune activation-related transcriptional and chromatin accessibility differences, as well as changes in the relative proportion of cardiomyocytes, neurons, and perivascular cells. In addition, we compare our adult-derived ATAC-Seq profiles to analogous fetal cell types to identify putative developmental-stage-specific regulatory factors. Finally, we train predictive models of cell-type-specific RNA expression levels utilizing ATAC-Seq profiles to link distal regulatory sequences to promoters, quantifying the predictive value of a simple TF-to-expression regulatory grammar and identifying cell-type-specific TFs.

genomics↗

bioRxiv2022

Substrate topology and cognate immunity modulate antibacterial toxicity of Tae cell wall hydrolases

Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

biochemistry↗