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High affinity chromodomains engineered for improved detection of histone modifications and enhanced CRISPR-based gene repression

ABSTRACT: High affinity chromodomains engineered for improved detection of histone modifications and enhanced CRISPR-based gene repression

PROVIDER: PRJNA778779 | ENA |

REPOSITORIES: ENA

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High affinity chromodomains engineered for improved detection of histone modifications and enhanced CRISPR-based gene repression

Project description:Histone methylation is an important post-translational modification that plays a crucial role in regulating cellular functions, and its dysregulation is implicated in cancer and developmental defects. Therefore, systematic characterization of histone methylation is necessary to elucidate complex biological processes, identify biomarkers, and ultimately, enable drug discovery. Studying histone methylation relies on the use of antibodies, but these suffer from batch-to-batch variation, are costly, and cannot be used in live cells. Chromatin-modification reader domains are potential affinity reagents for methylated histones, but their application is limited by their modest affinities. We used phage display to identify key residues that greatly enhance the affinities of Cbx chromodomains for methylated histone marks and developed a general strategy for enhancing the affinity of most chromodomains of the human Cbx protein family. Our strategy allowed us to develop powerful probes for genome-wide binding analysis and live cell imaging. Furthermore, we used optimized chromodomains to develop extremely potent CRISPR-based repressors for tailored gene silencing. Our results highlight the power of engineered chromodomains for analyzing protein interaction networks involving chromatin and represent a modular platform for efficient gene silencing.

2022-09-08 | GSE188439 | GEO

High-affinity chromodomains engineered for improved detection of histone methylation and enhanced CRISPR-based gene repression.

Project description:Histone methylation is an important post-translational modification that plays a crucial role in regulating cellular functions, and its dysregulation is implicated in cancer and developmental defects. Therefore, systematic characterization of histone methylation is necessary to elucidate complex biological processes, identify biomarkers, and ultimately, enable drug discovery. Studying histone methylation relies on the use of antibodies, but these suffer from lot-to-lot variation, are costly, and cannot be used in live cells. Chromatin-modification reader domains are potential affinity reagents for methylated histones, but their application is limited by their modest affinities. We used phage display to identify key residues that greatly enhance the affinities of Cbx chromodomains for methylated histone marks and develop a general strategy for enhancing the affinity of chromodomains of the human Cbx protein family. Our strategy allows us to develop powerful probes for genome-wide binding analysis and live-cell imaging. Furthermore, we use optimized chromodomains to develop extremely potent CRISPR-based repressors for tailored gene silencing. Our results highlight the power of engineered chromodomains for analyzing protein interaction networks involving chromatin and represent a modular platform for efficient gene silencing.

| S-EPMC9666628 | biostudies-literature

Improved methods for deamination-based m6A detection

Project description:We present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced m6A recognition in APO1-YTH (D422N). In addition, we develop in vitro DART-seq and show that it performs similarly to cellular DART-seq and can map m6A in any sample of interest using nanogram amounts of total RNA.

2022-05-11 | GSE196416 | GEO

Characterization of RNAs captured through a new engineered protein-mediated affinity purification

Project description:Characterization of RNAs captured through a new engineered protein-mediated affinity purification

| PRJNA884313 | ENA

Characterization of RNAs captured through a new engineered protein-mediated affinity purification

Project description:We generated a new recombinant protein HARD (High-affinity RNA-binding Domain) with non-sequence-specific high-affinity on RNAs and developed the immobilized-HARD protein-mediated RNA affinity purification approach (HARD-AP).We showed that HARD-AP successfully captured all RNA biotypes.

2024-08-23 | GSE214173 | GEO

Improved methods for deamination-based m6A detection

Project description:Improved methods for deamination-based m6A detection

| PRJNA804865 | ENA

Transcriptomic analysis of engineered and improved Pichia pastoris for S-adenosyl-l-methionine (SAM) production

Project description:SAM is the principal methyl group donor in all living organisms, and has attracted much interest in clinical research. We improved SAM production in engineered P. pastoris strain GS115/DS56 by overexpression of the recombinant methionine adenosyltransferase (MAT) gene DS56, and further enhanced SAM prduction in G12-CBS by downregulation of the cystathionine β-synthase (CBS) gene CYS4 with a weak promoter G12. We performed the pairwise transcriptome comparisons between high-producing (HP) and low-producing (LP) strains, in order to understand the impact of SAM accumulation on the P. pastoris physiology and to identify genome-wide targets for further improving SAM production.

2018-09-01 | GSE114152 | GEO

The enhanced functionality of low-affinity CD19 CAR T cells is associated with activation priming and a polyfunctional cytokine phenotype

Project description:We have compared a low-affinity second-generation anti-CD19 CAR (CAT) with the high-affinity FMC63 CAR used in tisagenlecleucel to understand the unknown molecular mechanisms that drive different CAR T cell treatment outcomes. CAT cells are characterized by a faster antigen dissociation rate and present an enhanced in vivo expansion and anti-tumor efficacy, showing lower toxicity to patients and a long-term persistence in a Phase I clinical study. To understand this improved CAT cells behaviour, we performed a systematic in vitro characterization of the transcriptomic (RNA-seq) and protein (CyTOF) changes between the two groups. The analysis focused on T cells expressing low-affinity vs high-affinity anti-CD19 CARs following stimulation with CD19-expressing cells. Our results show that CAT potent and long-term anti-tumour responses may be sustained by the establishment of a CAR T cells self-reinforcing circuit activated through cytokines polyfunctional crosstalk. This study may inform the design of versatile CAR T cells, capable of balancing safety and efficacy.

2022-11-22 | GSE157584 | GEO

Transcriptomic analysis of engineered and improved Pichia pastoris for S-adenosyl-l-methionine (SAM) production

Project description:Transcriptomic analysis of engineered and improved Pichia pastoris for S-adenosyl-l-methionine (SAM) production

| PRJNA464437 | ENA

Enhanced detection of RNA modifications with high-accuracy nanopore RNA basecalling models

Project description:Chemical RNA modifications, collectively referred to as the ‘epitranscriptome’, have been intensively studied during the last years, largely facilitated by the use of next-generation sequencing technologies. Recent efforts have turned towards the nanopore direct RNA sequencing (DRS) platform, as it allows simultaneous detection of diverse RNA modification types in full-length native RNA molecules. While RNA modifications can be identified in the form of systematic basecalling ‘errors’ in DRS datasets, m6A modifications produce very modest ‘errors’, limiting the applicability of this approach to sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully-unmodified in vitro synthetic sequences, increase the ‘error’ signal of m6A modifications, leading to enhanced detection of RNA modifications even at lower stoichiometries. We then show that the use of these models enhances the detection of RNA modifications on previously published in vivo human samples, using third-party softwares for the detection of RNA modifications. Moreover, our work provides a novel RNA basecalling model that shows a median accuracy of 97%, compared to previously available RNA basecalling models that show 91% accuracy. Notably, this increase in accuracy does not only lead to improved detection of RNA modifications, but also enhanced mappability of RNA reads, which becomes more evident in the case of short RNA reads (50% increase). Altogether, our work stresses the importance of using fully unmodified RNA sequences for training RNA basecalling models, and how the use of different basecalling models can significantly affect the detection of RNA modifications and read mappability.

2024-08-26 | GSE246151 | GEO

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