Recently, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBEs have the significant liabilities of off-target and bystander editing activities, partly due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study, we engineered optimized, soluble and stable Cas-embedded CBEs (CE_CBEs) that integrate several recent advances, which were efficiently formulated for direct delivery into cells as ribonucleoprotein (RNP) complexes.
View Article and Find Full Text PDFGene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers.
View Article and Find Full Text PDFEditing the +58 region of the BCL11A erythroid enhancer has shown promise in treating β-globin disorders. To address variations in fetal hemoglobin (HbF) response, we investigated editing both +58 and +55 enhancers. Rhesus macaques transplanted with edited hematopoietic stem/progenitor cells (HSPCs) following busulfan conditioning exhibited durable, high-level (∼90%) editing frequencies post transplantation with sustained HbF reactivation over 4 years, without hematological perturbations.
View Article and Find Full Text PDFBackground: Decades of evidence have elucidated associations between early adversity and risk for negative outcomes. However, traditional conceptualizations of the biologic embedding of adversity ignore neuroscientific principles which emphasize developmental plasticity. Dimensional models suggest that separate dimensions of experiences shape behavioral development differentially.
View Article and Find Full Text PDFObjective: To test whether pediatrician training leads to provider utilization of stimulant diversion prevention strategies as reported by adolescent patients with ADHD.
Methods: Pediatric practices received a stimulant diversion prevention workshop (SDP) or continued treatment-as-usual (TAU) in a cluster-randomized controlled trial. Surveys were completed by 341 stimulant-treated patients at baseline and three follow-up assessments.
CRISPR tiling screens have advanced the identification and characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing. This study introduces , an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. This approach enables the dissection of regulatory elements at nucleotide resolution, facilitating a direct assessment of genotype-phenotype effects.
View Article and Find Full Text PDFThere have been long and bitter debates between those who advocate for the use of residualized change as the foundation of longitudinal models versus those who utilize difference scores. However, these debates have focused primarily on modeling change in the outcome variable. Here, we extend these same ideas to the covariate side of the change equation, finding similar issues arise when using lagged versus difference scores as covariates of interest in models of change.
View Article and Find Full Text PDFGenome editing with RNA-guided DNA binding factors carries risk of off-target editing at homologous sequences. Genetic variants may introduce sequence changes that increase homology to a genome editing target, thereby increasing risk of off-target editing. Conventional methods to verify candidate off-targets rely on access to cells with genomic DNA carrying these sequences.
View Article and Find Full Text PDFIn analyzing longitudinal data with growth curve models, a critical assumption is that changes in the observed measures reflect construct changes and not changes in the manifestation of the construct over time. However, growth curve models are often fit to a repeated measure constructed as a sum or mean of scale items, making an implicit assumption of constancy of measurement. This practice risks confounding actual construct change with changes in measurement (i.
View Article and Find Full Text PDFModern medical moulages are becoming increasingly important in simulation-based health professions education. Their lifelikeness is important so that simulation engagement is not disrupted while their standardization is crucial in high-stakes exams. This report describes in detail how three-dimensional transfers are developed and produced so that educators will be able to develop their own.
View Article and Find Full Text PDFPrime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La.
View Article and Find Full Text PDFThe YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored.
View Article and Find Full Text PDFEx vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin.
View Article and Find Full Text PDFBackground: Correct individual tonotopic frequency stimulation of the cochlea plays an important role in the further development of anatomy-based cochlear implantation. In this context, frequency-specific fitting of the basal electrode contact with a normal insertion depth can be difficult since it is often placed in a frequency range higher than 10 kHz, and current audio processors only stimulate for frequencies up to 8.5 kHz due to microphone characteristics.
View Article and Find Full Text PDFOver the last 5 years, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBE's have the significant liabilities of off-target and bystander editing activities, in part due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study we engineeredhighly stabilized Cas-embedded CBEs (sCE_CBEs) that integrate several recent advances, andthat are highly expressible and soluble for direct delivery into cells as ribonucleoprotein (RNP) complexes.
View Article and Find Full Text PDFAs our understanding of the tumor microenvironment grows, the pathology field is increasingly utilizing multianalyte diagnostic assays to understand important characteristics of tumor growth. In clinical settings, brightfield chromogenic assays represent the gold-standard and have developed significant trust as the first-line diagnostic method. However, conventional brightfield tests have been limited to low-order assays that are visually interrogated.
View Article and Find Full Text PDFAlcohol use among Biracial adolescents remains understudied. This study examined how parenting and peer factors relate to age of alcohol use onset among Black, White, and Biracial Black-White adolescents and emerging adults. We used Add Health data to produce a final analytic sample of 13,528 adolescents who self-identified as White, Black, or Biracial Black-White.
View Article and Find Full Text PDFThe cochlear aqueduct (CA), which connects the scala tympani and the subarachnoid space, and its accompanying structures appear to have a significant relevance during cochlear implantation and an accurate visualization in clinical imaging is of great interest. This study aims to determine which potential and limitations clinically available imaging modalities have in the visualization of the CA. Micro-CT, flat-panel volume computed tomography with and without secondary reconstruction (fpVCT, fpVCTseco) and multislice computed tomography (MSCT) of 10 temporal bone specimen were used for 3D analysis of the CA.
View Article and Find Full Text PDFGene regulatory networks (GRNs) are key determinants of cell function and identity and are dynamically rewired during development and disease. Despite decades of advancement, challenges remain in GRN inference, including dynamic rewiring, causal inference, feedback loop modeling and context specificity. To address these challenges, we develop Dictys, a dynamic GRN inference and analysis method that leverages multiomic single-cell assays of chromatin accessibility and gene expression, context-specific transcription factor footprinting, stochastic process network and efficient probabilistic modeling of single-cell RNA-sequencing read counts.
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