RESUMO
Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.
Assuntos
Citidina Desaminase , Edição de Genes , Humanos , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/metabolismo , Dedos de Zinco , Citidina/genética , Sistemas CRISPR-CasRESUMO
Predicting the function of noncoding variation is a major challenge in modern genetics. In this study, we used massively parallel reporter assays to screen 5706 variants identified from genome-wide association studies for both Alzheimer's disease (AD) and progressive supranuclear palsy (PSP), identifying 320 functional regulatory variants (frVars) across 27 loci, including the complex 17q21.31 region. We identified and validated multiple risk loci using CRISPR interference or excision, including complement 4 (C4A) and APOC1 in AD and PLEKHM1 and KANSL1 in PSP. Functional variants disrupt transcription factor binding sites converging on enhancers with cell type-specific activity in PSP and AD, implicating a neuronal SP1-driven regulatory network in PSP pathogenesis. These analyses suggest that noncoding genetic risk is driven by common genetic variants through their aggregate activity on specific transcriptional programs.
Assuntos
Doença de Alzheimer , Cromossomos Humanos Par 17 , Redes Reguladoras de Genes , Variação Genética , Regiões não Traduzidas , Doença de Alzheimer/genética , Cromossomos Humanos Par 17/genética , Genes Reporter , Loci Gênicos , Estudo de Associação Genômica Ampla , Humanos , Fatores de Risco , Paralisia Supranuclear Progressiva/genética , Regiões não Traduzidas/genéticaRESUMO
A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.
Assuntos
Isopropiltiogalactosídeo/farmacologia , Lógica , Regiões Promotoras Genéticas , Sítios de Ligação , Fenômenos Biofísicos , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Fluorescência , Genes Reporter , Mutação/genética , Regiões Operadoras Genéticas/genética , Ligação Proteica , Reprodutibilidade dos Testes , Termodinâmica , Fatores de Transcrição/metabolismoRESUMO
In eukaryotes, transcription factors (TFs) orchestrate gene expression by binding to TF-binding sites (TFBSs) and localizing transcriptional co-regulators and RNA polymerase II to cis-regulatory elements. However, we lack a basic understanding of the relationship between TFBS composition and their quantitative transcriptional responses. Here, we measured expression driven by 17,406 synthetic cis-regulatory elements with varied compositions of a model TFBS, the c-AMP response element (CRE) by using massively parallel reporter assays (MPRAs). We find CRE number, affinity, and promoter proximity largely determines expression. In addition, we observe expression modulation based on the spacing between CREs and CRE distance to the promoter, where expression follows a helical periodicity. Finally, we compare library expression between an episomal MPRA and a genomically integrated MPRA, where a single cis-regulatory element is assayed per cell at a defined locus. These assays largely recapitulate each other, although weaker, non-canonical CREs exhibit greater activity in a genomic context.
Assuntos
Monofosfato de Adenosina/metabolismo , Genômica/métodos , Plasmídeos/metabolismo , Elementos de Resposta/genética , HumanosRESUMO
Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder. Large genetically informative cohorts of individuals with ASD have led to the identification of a limited number of common genome-wide significant (GWS) risk loci to date. However, many more common genetic variants are expected to contribute to ASD risk given the high heritability. Here, we performed a genome-wide association study (GWAS) on 6222 case-pseudocontrol pairs from the Simons Foundation Powering Autism Research for Knowledge (SPARK) dataset to identify additional common genetic risk factors and molecular mechanisms underlying risk for ASD. We identified one novel GWS locus from the SPARK GWAS and four significant loci, including an additional novel locus from meta-analysis with a previous GWAS. We replicated the previous observation of significant enrichment of ASD heritability within regulatory regions of the developing cortex, indicating that disruption of gene regulation during neurodevelopment is critical for ASD risk. We further employed a massively parallel reporter assay (MPRA) and identified a putative causal variant at the novel locus from SPARK GWAS with strong impacts on gene regulation (rs7001340). Expression quantitative trait loci data demonstrated an association between the risk allele and decreased expression of DDHD2 (DDHD domain containing 2) in both adult and prenatal brains. In conclusion, by integrating genetic association data with multi-omic gene regulatory annotations and experimental validation, we fine-mapped a causal risk variant and demonstrated that DDHD2 is a novel gene associated with ASD risk.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Transtorno do Espectro Autista/genética , Estudo de Associação Genômica Ampla , Humanos , Fosfolipases , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Fatores de RiscoRESUMO
The >800 human G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state- and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here, we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G protein signal transduction. We tested 7800 of 7828 possible single amino acid substitutions to the beta-2 adrenergic receptor (ß2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for ß2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we identify residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.
Assuntos
Análise Mutacional de DNA/métodos , Receptores Acoplados a Proteínas G/química , Clonagem Molecular , Código de Barras de DNA Taxonômico , Edição de Genes , Células HEK293 , Humanos , Aprendizado de Máquina , Modelos Moleculares , Conformação Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The mitotic apparatus of the early sea urchin embryo is the archetype example of a centrosome-dominated, large aster spindle organized by means of the centriole of the fertilizing sperm. In this study, we tested the hypothesis that artificially activated sea urchin eggs possess the capacity to assemble the anastral, bipolar spindles present in many acentrosomal systems. Control fertilized Lytechinus pictus embryos and ammonia-activated eggs were immunolabeled for tubulin, centrosomal material, the spindle pole structuring protein NuMA and the mitotic kinesins MKLP1/Kinesin-6, Eg5/Kinesin-5, and KinI/Kinesin-13. Confocal imaging showed that a subset of ammonia-activated eggs contained bipolar "mini-spindles" that were anastral; displayed metaphase and anaphase-like stages; labeled for centrosomal material, NuMA, and the three mitotic kinesins; and were observed in living eggs using polarization optics. These results suggest that spindle structural and motor proteins have the ability to organize bipolar, anastral spindles in sea urchin eggs activated in the absence of the paternal centriole.