Base editing mutagenesis maps functional alleles to tune human T cell activity
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ABSTRACT:
PROVIDER: PRJNA1024532 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26297300.fastq.gz | Fastqsanger.gz | |||
| SRR26297301.fastq.gz | Fastqsanger.gz | |||
| SRR26297302.fastq.gz | Fastqsanger.gz | |||
| SRR26297303.fastq.gz | Fastqsanger.gz | |||
| SRR26297304.fastq.gz | Fastqsanger.gz |
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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: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.
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Project description:DNA base damage is an important contributor to genome instability, but how the formation and repair of these lesions is affected by the genomic landscape is unknown. Here we describe genome-wide maps of DNA base damage, repair, and mutagenesis at single nucleotide resolution in yeast treated with the alkylating agent methyl methanesulfonate (MMS). Analysis of these maps revealed that base excision repair (BER) of alkylation damage is significantly modulated by chromatin, with faster repair in nucleosome free regions, and slower repair and higher mutation density within strongly positioned nucleosomes. Both the translational and rotational settings of lesions within nucleosomes significantly influence BER efficiency; moreover, this effect is asymmetric relative to the nucleosome dyad and is regulated by histone modifications. Our data also indicate that MMS-induced A mutations are significantly enriched on the non-transcribed strand (NTS) of yeast genes, particularly in BER-deficient strains, due to higher damage formation on the NTS and transcription-coupled repair of the transcribed strand (TS). These findings reveal the influence of chromatin on repair and mutagenesis of base lesions on a genome-wide scale, and suggest a novel mechanism for transcription-associated mutation asymmetry, which is frequently observed in human cancers.
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Project description:We examined the off-targets of new base editing tools at RNA level
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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.
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Project description: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.
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Project description: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.
2024-01-12 | GSE253182 | GEO
Project description:Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [3H]thymidine into CHO hamster cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular global response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [3H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC>CG and AT>CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses.
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Project description:Efficient CRISPR-mediated base editing in Agrobacterium spp.
| PRJEB38304 | ENA