Biology subjects

Conklin, B. R.

Publications and source records attributed to Conklin, B. R..

18 recordsLinked to original sources

bioRxiv2026

EXCAVATE-HT: A Bioinformatic Pipeline to Identify Targetable Genomic Variants for Allele-Specific Editing

Allele-specific CRISPR/Cas editing is a powerful tool with great potential for treating genetic diseases and for uncovering the effects of allelic diversity. By targeting commonly inherited single nucleotide polymorphisms (SNPs), a small number of gRNAs can treat many more individuals than targeting rare disease mutations. However, current tools for identifying common targetable variants and generating CRISPR guide RNAs (gRNA) have fundamental conceptual and technical limitations. Here, we introduce EXCAVATE-HT (EXtracting Common Allelic VAriants for Targeted Editing in High-Throughput) a bioinformatic tool that mines population variant data to generate CRISPR libraries targeting genomic loci for allele-specific editing. Users define their loci of interest, Cas species, and SNP frequency, then EXCAVATE-HT outputs an annotated list of allele-specific gRNAs. EXCAVATE-HT can also generate libraries of gRNA pairs to enable excision. We illustrate the use of EXCAVATE-HT to design and characterize multiple gRNA libraries for allele-specific targeting of the disease gene, Cone-Rod Homeobox (CRX). EXCAVATE-HT revealed multiple excisions that could treat >30-fold more patients than targeting a single CRX disease mutation.

bioinformatics↗

bioRxiv2026

Using image classifiers to predict CMT2A disease-relevant mitochondrial motility phenotypes in iPSC motor neurons

Charcot-Marie-Tooth disease type 2A (CMT2A) is a genetic disease characterized by autosomal dominant MFN2 mutations and dysregulated mitochondrial trafficking. While there is currently no FDA-approved CMT2A therapy, the recent development of iPSC motor neuron model systems, high-throughput imaging platforms, and CRISPR-based gene editing technologies holds promise for screening new therapies at scale in vitro. A critical roadblock for therapeutic screening is the development of scalable and robust computational methods to assess the mitochondrial trafficking phenotypes, healthy or diseased, of each iPSC motor neuron sample. To address this gap, we developed a vision transformer (ViT) based classification framework that predicts disease phenotypes using kymographs, an image transformation that captures particle movement along a prespecified path, such as mitochondrial movement along axons. We show that our classification approach more accurately discriminates healthy MFN2 wild-type (WT) from diseased MFN2 R364W-mutant (R364W) iPSCs than alternative summary statistics, such as mitochondrial speed and fraction of stationary mitochondria that are directly extracted from kymographs. Furthermore, we show that our model maintains high accuracy when deployed on a biological replicate holdout dataset. An analysis of ViT patch embeddings of the kymographs shows that mitochondria with highly variable sizes and many intersection events most strongly associate with R364W diseased kymographs. The computational approach demonstrated in this paper has broad applicability for future high-throughput screens where organelle trafficking along axons plays a key role in disease pathogenesis.

cell biology↗

bioRxiv2024

Haplotype editing with CRISPR/Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL

Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplo-sufficient. We previously edited a dominant NEFL missense mutation with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different NEFL missense mutations cause disease. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with NEFL disease mutations for gene excision. We validated this haplotype editing approach for two different missense mutations and demonstrated its therapeutic potential in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent byproduct of excision editing, failed to reliably disrupt mutant allele expression. We deployed alternative strategies and novel molecular assays to increase therapeutic editing outcomes while maintaining specificity for the mutant allele. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.

genetics↗

bioRxiv2024

Programmable epigenome editing by transient delivery of CRISPR epigenome editor ribonucleoproteins

Programmable epigenome editors modify gene expression in mammalian cells by altering the local chromatin environment at target loci without inducing DNA breaks. However, the large size of CRISPR-based epigenome editors poses a challenge to their broad use in biomedical research and as future therapies. Here, we present Robust ENveloped Delivery of Epigenome-editor Ribonucleoproteins (RENDER) for transiently delivering programmable epigenetic repressors (CRISPRi, DNMT3A-3L-dCas9, CRISPRoff) and activator (TET1-dCas9) as ribonucleoprotein complexes into human cells to modulate gene expression. After rational engineering, we show that RENDER induces durable epigenetic silencing of endogenous genes across various human cell types, including primary T cells. Additionally, we apply RENDER to epigenetically repress endogenous genes in human stem cell-derived neurons, including the reduction of the neurodegenerative disease associated V337M-mutated Tau protein. Together, our RENDER platform advances the delivery of CRISPR-based epigenome editors into human cells, broadening the use of epigenome editing in fundamental research and therapeutic applications.

genetics↗

bioRxiv2024

Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

molecular biology↗

bioRxiv2023

Dual α-globin and truncated erythropoietin receptor knock-in restores hemoglobin production in α-thalassemia major-derived hematopoietic stem and progenitor cells

Alpha-thalassemia is an autosomal recessive disease with increasing worldwide prevalence. The molecular basis is due to mutation or deletion of one or more duplicated -globin genes, and disease severity is directly related to the number of allelic copies compromised. The most severe form, -thalassemia major (TM), results from loss of all four copies of -globin and has historically resulted in fatality in utero. However, in utero transfusions now enable survival to birth. Postnatally, patients face challenges similar to {beta}-thalassemia, including severe anemia and erythrotoxicity due to imbalance of {beta}-globin and -globin chains. While curative, hematopoietic stem cell transplantation (HSCT) is limited by donor availability and potential transplant-related complications. Despite progress in genome editing treatments for {beta}-thalassemia, there is no analogous curative option for patients suffering from -thalassemia. To address this, we designed a novel Cas9/AAV6-mediated genome editing strategy that integrates a functional -globin gene into the {beta}-globin locus in TM patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the -globin integration cassette dramatically increased erythropoietic output from edited HSPCs and led to the most robust production of -globin, and consequently normal hemoglobin. By directing edited HSPCs toward increased production of clinically relevant RBCs instead of other divergent cell types, this approach has the potential to mitigate the limitations of traditional HSCT for the hemoglobinopathies, including low genome editing and low engraftment rates. These findings support development of a definitive ex vivo autologous genome editing strategy that may be curative for -thalassemia. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/555926v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1b63dacorg.highwire.dtl.DTLVardef@18b25a1org.highwire.dtl.DTLVardef@53835corg.highwire.dtl.DTLVardef@d52bf5_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Two therapeutic CRISPR/Cas9 gene editing approaches revert FTD/ALS cellular pathology caused by a C9orf72 repeat expansion mutation in patient derived cells

CRISPR gene editing holds promise to cure or arrest genetic disease, if we can find and implement curative edits reliably, safely and effectively. Expansion of a hexanucleotide repeat in C9orf72 is the leading known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We evaluated three approaches to editing the mutant C9orf72 gene for their ability to correct pathology in neurons derived from patient iPSCs: excision of the repeat region, excision of the mutant allele, and excision of regulatory region exon 1A. All three approaches normalized RNA abnormalities and TDP-43 pathology, but only repeat excision and mutant allele excision completely eliminated pathologic dipeptide repeats. Our work sheds light on the complex regulation of the C9orf72 gene and suggests that because of sense and anti-sense transcription, silencing a single regulatory region may not reverse all pathology. Our work also provides a roadmap for evaluating CRISPR gene correction using patient iPSCs.

neuroscience↗

bioRxiv2021

Functional analysis of a common BAG3 allele associated with protection from heart failure

Multiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism for such protection remains elusive. One of the variants in this allele block is coding, changing cysteine to arginine at position 151 of BAG3 (rs2234962-BAG3C151R). Here, we use induced pluripotent stem cells (iPSC) to test if the BAG3C151R variant alters protein and cellular function in human cardiac myocytes. Quantitative protein interaction network analysis identified specific changes in BAG3C151R protein interaction partners in cardiomyocytes but not in iPSCs or an immortalized cell line. Knockdown of BAG3 interacting factors in cardiomyocytes followed by myofibrillar analysis revealed that BAG3C151R associates more strongly with proteins involved in the maintenance of myofibrillar integrity. Finally, we demonstrate that cardiomyocytes expressing the BAG3C151R variant have improved response to proteotoxic stress in an allele dose-dependent manner. This study suggests that the BAG3C151R variant increases cardiomyocyte protection from stress by enhancing the recruitment of factors critical to the maintenance of myofibril integrity, hinting that this variant could be responsible for the cardioprotective effect of the haplotype block. By revealing specific changes in preferential binding partners of the BAG3C151R protein variant, we also identify potential targets for the development of novel cardioprotective therapies.

genetics↗

bioRxiv2021

Allele-specific gene editing rescues pathology in a human model of Charcot-Marie-Tooth disease type 2E

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.

genetics↗

bioRxiv2021

Synchronization of iPS-derived cardiomyocytes to visitor heartbeat in an interactive museum exhibit

Science museums play an important role in science education, both engaging the public with science concepts and building support for scientific research. Designing museum exhibits to meet increasing public interests in the life sciences is particularly important, yet remains challenging. In this report we describe Give Heart Cells a Beat, a permanent interactive science exhibit that allows museum visitors to synchronize the beating of live stem cell-derived cardiomyocytes to their own heart rate in real-time. Evaluation with museum visitors reveals that the exhibit engaged the public with the specimen and prompted curiosity in heart biology and, to a lesser degree, stem cells and electrophysiology. Give Heart Cells a Beat is the product of a close collaboration between a museum and a research laboratory, and, to our knowledge, the first example of the use of live human heart cells in an interactive exhibit. We hope this exhibit will serve as an example for the implementation of stem cell technology in the field of informal science education and encourage others to pursue close working relationships between academia and public science venues such as museums.

scientific communication and education↗

bioRxiv2021

Integration of Protein Interactome Networks with Congenital Heart Disease Variants Reveals Candidate Disease Genes

Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains a challenge despite large-scale genomic sequencing efforts. We hypothesized that genetic determinants for CHDs may lie in protein interactomes of GATA4 and TBX5, two transcription factors that cause CHDs. Defining their interactomes in human cardiac progenitors via affinity purification-mass spectrometry and integrating results with genetic data from the Pediatric Cardiac Genomic Consortium revealed an enrichment of de novo variants among proteins that interact with GATA4 or TBX5. A consolidative score that prioritized interactome members based on variant, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied cardiac developmental genes, resulting in co-activation, and the GLYR1 missense variant associated with CHD disrupted interaction with GATA4. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating the contribution of genetic variants in disease.

genetics↗

bioRxiv2020

uPTI: uniaxial permittivity tensor imaging of intrinsic density and anisotropy

Biological architecture is intrinsically tensorial. The permittivity tensor (PT) of biological material reports the density, angular anisotropy, symmetry, and 3D orientation of biomolecules. High-resolution measurement of PT can enable quantitative and label-free analysis of organelle, cell, and tissue architecture, but remains challenging. We report uniaxial permittivity tensor imaging (uPTI), a label-free computational imaging method for volumetric measurement of PT with diffraction-limited resolution. uPTI encodes the components of PT into intensity modulations using oblique illumination and polarization-resolved imaging. The high-dimensional data is decoded with a vectorial image formation model and a multi-channel convex optimization, assuming that the molecular distribution in each voxel has uniaxial symmetry. We describe a modular implementation of uPTI that can be multiplexed with complementary imaging modalities. We report volumes of uPT in mouse brain tissue, SARS-CoV-2 infected cardiomyocytes, RSV infected A549 cells, H&E stained tissue sections, isotropic beads, and anisotropic glass targets. uPTI enabled volumetric imaging of the 3D orientation and symmetry of organelles, cells, and tissue components with higher spatio-angular resolution than current vectorial tomography, ptychography, and light-field microscopy methods. We provide an open source implementation of the image formation model and reconstruction algorithms.

biophysics↗

bioRxiv2020

Silencing of E-cadherin in induced human pluripotent stem cells promotes extraembryonic fates accompanying multilineage differentiation

Summary/AbstractIn embryonic development, symmetry breaking events and the mechanical milieus in which they occur coordinate the specification of separate cell lineages. Here, we use 3D aggregates of human pluripotent stem cells (hPSCs) encapsulated in alginate microbeads to model the early blastocyst prior to zona pellucida hatching. We demonstrate that 3D confinement combined with modulation of cell-cell adhesions is sufficient to drive differentiation and collective migration reminiscent of the pre-implantation embryo. Knockdown of the cell adhesion protein CDH1 in encapsulated hPSC aggregates resulted in protrusion morphologies and emergence of extra-embryonic lineages, whereas unencapsulated CDH1(-) aggregates displayed organized radial delamination and mesendoderm specification bias. Transcriptomic similarities between single-cell RNA-sequencing data of early human embryos and encapsulated CDH1(-) aggregates establishes this in vitro system as a competent surrogate for studying early embryonic fate decisions and highlights the relationship between cell-cell adhesions and the mechanical microenvironment in directing cell fate and behavior. HighlightsO_LIGeneration of embryonic scale 3D morphogenesis using hydrogel encapsulation C_LIO_LIManipulating adhesion triggers emergence of specific morphologies and cell fates C_LIO_LIAcquisition of germ layer cell fates mimics early human embryonic diversity C_LI

developmental biology↗

bioRxiv2020

SARS-CoV-2 infection of human iPSC-derived cardiac cells predicts novel cytopathic features in hearts of COVID-19 patients

Although COVID-19 causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human iPSC-derived heart cells to SARS-CoV-2 revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural proteins corroborated adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and numerous iPSC-cardiomyocytes lacking nuclear DNA. Human autopsy specimens from COVID-19 patients displayed similar sarcomeric disruption, as well as cardiomyocytes without DNA staining. These striking cytopathic features provide new insights into SARS-CoV-2 induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise serious concerns about the long-term consequences of COVID-19.

cell biology↗

bioRxiv2020

Rapid, precise quantification of large DNA excisions and inversions by ddPCR

The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (>20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.

molecular biology↗

bioRxiv2018

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides an unprecedented view of events that precede repair of the affected sites.

molecular biology↗

bioRxiv2018

Leveraging genetic variants for personalized and allele-specific sgRNA design

The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing by even a single base pair. However, current tools only design sgRNAs for a reference genome, not taking into account individual variants which may generate, remove, or modify CRISPR/Cas sgRNA sites. This may cause mismatches between designed sgRNAs and the individual genome they are intended to target, leading to decreased experimental performance. Here we describe AlleleAnalyzer, a tool for designing personalized and allele-specific sgRNAs for genome editing. We leverage >2,500 human genomes to identify optimized pairs of sgRNAs that can be used for human therapeutic editing in large populations in the future.

bioinformatics↗

bioRxiv2018

Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference

Morphogenesis results from the interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events offer little control over parallel cell type co-emergence and do not offer the capability to selectively perturb gene expression in specific subpopulations of cells. We have developed an in vitro system that can spatiotemporally interrogate cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined the effects of independently knocking down molecular regulators of cortical tension and cell-cell adhesion using inducible CRISPRi: Rho-associated kinase-1 (ROCK1) and E-cadherin (CDH1), respectively. Induced mosaic knockdown of ROCK1 or CDH1 in hiPSC populations resulted in differential patterning events within hiPSC colonies indicative of cell-driven population organization. Patterned colonies retained an epithelial phenotype and nuclear expression of pluripotency markers. Gene expression within each of the mixed populations displayed a transient wave of differential expression with induction of knockdown that stabilized in coordination with intrinsic pattern formation. Mosaic patterning of hiPSCs enables the genetic interrogation of emergent multicellular properties of pluripotent cells, leading to a greater mechanistic understanding of the specific molecular pathways regulating the dynamics of symmetry breaking events that transpire during developmental morphogenesis.\n\nSIGNIFICANCEHuman embryonic development entails a series of multicellular morphogenic events that lead to primitive tissue formation. Attempts to study human morphogenic processes experimentally have been limited due to divergence from model organisms and the inability of current human in vitro models to accurately control the coincident emergence of heterogeneous cell populations in the spatially controlled manner necessary for proper tissue structure. We developed a human induced pluripotent stem cell (iPSC) in vitro model that enables temporal control over the emergence of heterotypic subpopulations of cells. We examined mosaic knockdown of two target molecules to create predictable and robust cell-patterning events within hiPSC colonies. This method allows for dynamic interrogation of intrinsic cell mechanisms that initiate symmetry breaking events and provides direct insight(s) into tissue developmental principles.

bioengineering↗