Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs

ABSTRACT: Translating genetic findings for neurodevelopmental and psychiatric disorders (NPD) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, here we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop-codons (iSTOP) that lead to mRNA nonsense-mediated-decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 NPD genes. Using RNAseq, we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Interestingly, for three schizophrenia risk genes (SETD1A, TRIO, CUL1), despite the high efficiency of base editing, we only obtained heterozygous LoF alleles, suggesting their essential roles for cell growth. We replicated the reported neural phenotypes of SHANK3-haploinsufficiency and found CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.

ORGANISM(S): Homo sapiens

PROVIDER: GSE262442 | GEO | 2024/09/17

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

CRISPR-CLEAR: Nucleotide-Resolution Mapping of Regulatory Elements via Allelic Readout of Tiled Base Editing

Project description: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 CRISPR-CLEAR, 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.

2024-10-03 | GSE278581 | GEO

Enhanced genome-wide knockout screens in bacteria with CRISPR base editors

Project description:Genome-wide knockout or knockdown screens have become powerful tools for the investigation of genotype-to-phenotype relationships. In bacteria, these screens commonly rely on transcriptional repression by dCas9, gene knockouts through Cas9 editing or random transposon mutagenesis, but depending on the technique, suffer from incomplete gene silencing, low editing efficiencies or they require massive library sizes. Here, we take a distinct approach with base editing to introduce premature stop codons or mutate start codons in Escherichia coli using a ScCas9 nickase derived base editor (ScBE3) that exhibits flexible PAM recognition. We then derive guide design rules by applying machine learning to a gene essentiality screen conducted in E. coli. For further improvement, we combined base-editing with Cas9-induced cleavage of the unedited cell fraction. The efficiency of this dual system was validated through a screen of conditionally essential E. coli genes. This improved setup that decouples the gene editing from the screening leads to more efficient guide depletion and confirmed previously published conditionally essential genes. Overall, base editing represents a useful tool for genome-wide knockout screens in bacteria and will eventually enable genome-wide knockout screens in a broader range of bacterial species to study their diverse genetics.

2024-02-05 | GSE225335 | GEO

Base editing mutagenesis maps functional alleles to tune human T cell activity

Project description:CRISPR-enabled genetic screening is a powerful tool to discover genes that control T cell function and has nominated candidate target genes for immunotherapies1–6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could discover alleles that tune specific phenotypes. DNA base editors are powerful tools to introduce targeted mutations with high efficiency7,8. Here, we develop a large-scale base editing mutagenesis platform with the goal of pinpointing nucleotides encoding amino acid residues that tune primary human T cell activation responses. We generated a library of ~117,000 sgRNAs targeting base editors to protein coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We discovered a broad spectrum of alleles with variants encoding critical residues (in PIK3CD, VAV1, LCP2, PLCG1 and DGKZ and others), comprising both gain-of-function and loss-of-function mutations. We validated the functional effects of diverse alleles and further demonstrated that base edit hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed PAM requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base editing screens in primary immune cells provide biochemical insights with potential to accelerate immunotherapy design.

2023-10-11 | GSE244774 | GEO

Project description:base editing

| PRJNA859372 | ENA

Project description:base editing

| PRJNA1150906 | ENA

Project description:base editing

| PRJNA884754 | ENA

ABE8e adenine base editor precisely and efficiently corrects a COL7A1 nonsense mutation

Project description:Base editing introduces precise single-nucleotide edits in genomic DNA and has the potential to treat genetic diseases such as the blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates. Here, we use ABE8e, a recently evolved ABE, to correct primary RDEB fibroblasts harboring the recurrent RDEB nonsense mutation c.5047 C>T (p.Arg1683Ter) in exon 54 of COL7A1 and use a next generation sequencing workflow to interrogate post-treatment outcomes. Electroporation of ABE8e mRNA into a bulk population of RDEB patient fibroblasts resulted in remarkably efficient (94.6%) correction of the pathogenic allele, restoring COL7A1 mRNA and expression of C7 protein in western blots and in 3D skin constructs. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. Taken together, we have established a highly efficient pipeline for gene correction in primary fibroblasts with a favorable safety profile. This work lays a foundation for developing therapies for RDEB patients using ex vivo or in vivo base editing strategies.

2022-08-25 | GSE211876 | GEO

DeepC: Predicting chromatin interactions using megabase scaled deep neural networks and transfer learning (Tiled-C)

Project description:Understanding 3D genome structure requires high throughput, genome-wide approaches. However, assays for all vs. all chromatin interaction mapping are expensive and time consuming, which severely restricts their usage for large-scale mutagenesis screens or for mapping the impact of sequence variants. Computational models sophisticated enough to grasp the determinants of chromatin folding provide a unique window into the functional determinants of 3D genome structure as well as the effects of genome variation. A chromatin interaction predictor should work at the base pair level but also incorporate large-scale genomic context to simultaneously capture the large scale and intricate structures of chromatin architecture. Similarly, to be a flexible and generalisable approach it should also be applicable to data it has not been explicitly trained on. To develop a model with these properties, we designed a deep neuronal network (deepC) that utilizes transfer learning to accurately predict chromatin interactions from DNA sequence at megabase scale. The model generalizes well to unseen chromosomes and works across cell types, Hi-C data resolutions and a range of sequencing depths. DeepC integrates DNA sequence context on an unprecedented scale, bridging the different levels of resolution from base pairs to TADs. We demonstrate how this model allows us to investigate sequence determinants of chromatin folding at genome-wide scale and to predict the importance of regulatory elements and the impact of sequence variations.

2020-07-08 | GSE137436 | GEO

CRISPR base editing of cis-regulatory elements enables target gene perturbations

Project description:CRISPR technology has demonstrated broad utility for controlling target gene expression. However, there remains a need for strategies capable of modulating expression via the precise editing of non-coding regulatory elements. Here we demonstrate that CRISPR base editors, a class of gene-modifying proteins capable of inducing single-base substitutions in DNA, can be harnessed to perturb target gene expression via the targeted mutagenesis of cis-acting sequences. Using the promoter region of the human huntingtin (HTT) gene as an initial target, we show that editing of the binding site for the transcription factor NF-κB led to a marked reduction in HTT gene expression. We find that these gene perturbations were persistent and specific, as a transcriptome-wide RNA-seq analysis revealed virtually no off-target effects. We further show that this base-editing platform could lower HTT in vivo, as its delivery to a mouse model of Huntington’s disease decreased HTT expression in striatal neurons, an outcome that we show also increased survival. Finally, we use this approach to target the amyloid-beta precursor protein, demonstrating that multiplexed editing of its promoter region could significantly perturb its expression, supporting the applicability of this method. These findings thus demonstrate the potential for base editors to regulate target gene expression.

2022-08-24 | GSE204833 | GEO

Base editing mutagenesis maps functional alleles to tune human T cell activity

Project description:Base editing mutagenesis maps functional alleles to tune human T cell activity

| PRJNA1024532 | ENA