Ontology highlight
ABSTRACT:
PROVIDER: PRJNA913593 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off- target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by “tiptoeing” over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of genome editing procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.
2020-12-08 | GSE153471 | GEO
Project description:Widespread genetic epistasis among cancer genes.
| PRJNA253627 | ENA
Project description:Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow rich phenotyping through single-cell RNA sequencing readouts, and separately, characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.
2023-05-02 | GSE215253 | GEO
Project description:High-throughput yeast CRISPR genome editing
| PRJEB23616 | ENA
Project description:The mammalian genome possesses a network of non-coding regulatory elements with key genes regulated by many enhancers. It remains unknown why these genes require multiple enhancers for regulation and what is the functional role of each enhancer contributing to a coordinated enhancer network. Here we develop a novel framework, named SEER (Systematic Enhancer Epistasis Regulatory network analysis), which leverages a suite of perturbative, mapping, and imaging approaches, combined with machine learning and Genome-Wide Association Study (GWAS) analysis to systematically and quantitatively anatomize the enhancer epistasis network. Applying the framework to the MYC locus, we revealed a hierarchical two-layer epistasis model and defined a class of synergistic regulatory elements (SREs) which can maintain both high expression and robustness upon perturbation. Via machine learning, we identified and validated that two features, spatial contacts and BRD4 coactivator condensation, are major factors in maintaining the synergistic interactions of SREs. We used SEER to predict the synergistic functions of non-coding variants in SREs for their clinical risks in cancer and autoimmune disorders. The SEER framework provides a novel approach and theory for delineating roles of the massive enhancer network in gene regulation and interpreting non-coding variants for clinical risks in complex diseases.
2022-08-12 | GSE160768 | GEO
Project description:Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically-relevant nucleases, SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis, we identified putative immunogenic epitopes on each nuclease. Then, we used computational modeling to rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants were substantially less recognized by adaptive immune components, including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses, while maintaining wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 was comparable in efficiency to wild-type SaCas9, but significantly reduced undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection.
2024-11-07 | PXD054579 | Pride