Similar Datasets
Project description:Programmable epigenetic editors that achieve long-term gene expression modulation hold immense therapeutic potential. However, the large size of current CRISPR-based epigenome editors has limited their widespread application. This study describes the engineering of compact, mRNA-delivered epigenetic repressors, termed CRISPR OFF-EE, using SpyCas9, intein-based split-SpyCas9 or the smaller SF01 Cas variant, to achieve highly efficient and durable silencing of endogenous genes. We developed "superfolder" V2 mRNA constructs with optimized codon usage, UTRs, and nuclear localization signals, which enhanced mRNA stability, translational output, and LNP packaging. These CRISPR OFF-EE V2 editors demonstrated significantly improved and durable gene silencing across multiple targets in variable cell lines. Remarkably, a single intravenous administration of lipid nanoparticles (LNPs) encapsulating the optimized OFF-EE-V2 mRNA and selected sgRNAs targeting PCSK9 in mice led to an approximately 90% reduction in circulating PCSK9 protein and a 55% reduction in LDL-C levels, with effects persisting for at least 180 days. Whole-genome bisulfite sequencing confirmed the high specificity of these editors, with SF01-based editors exhibiting fewer off-target methylation events than SpCas9-based editors. The platform also demonstrated multiplexing capability and a favorable safety profile with liver-restricted activity. These findings establish a robust foundation for advancing in vivo therapeutics based on precise and durable epigenetic silencing using transiently delivered mRNA editors.
2025-06-20 | GSE299252 | GEO
Project description:Programmable epigenetic editors that achieve long-term gene expression modulation hold immense therapeutic potential. However, the large size of current CRISPR-based epigenome editors has limited their widespread application. This study describes the engineering of compact, mRNA-delivered epigenetic repressors, termed CRISPR OFF-EE, using SpyCas9, intein-based split-SpyCas9 or the smaller SF01 Cas variant, to achieve highly efficient and durable silencing of endogenous genes. We developed "superfolder" V2 mRNA constructs with optimized codon usage, UTRs, and nuclear localization signals, which enhanced mRNA stability, translational output, and LNP packaging. These CRISPR OFF-EE V2 editors demonstrated significantly improved and durable gene silencing across multiple targets in variable cell lines. Remarkably, a single intravenous administration of lipid nanoparticles (LNPs) encapsulating the optimized OFF-EE-V2 mRNA and selected sgRNAs targeting PCSK9 in mice led to an approximately 90% reduction in circulating PCSK9 protein and a 55% reduction in LDL-C levels, with effects persisting for at least 180 days. Whole-genome bisulfite sequencing confirmed the high specificity of these editors, with SF01-based editors exhibiting fewer off-target methylation events than SpCas9-based editors. The platform also demonstrated multiplexing capability and a favorable safety profile with liver-restricted activity. These findings establish a robust foundation for advancing in vivo therapeutics based on precise and durable epigenetic silencing using transiently delivered mRNA editors.
2025-06-20 | GSE299303 | GEO
Project description:mRNA-Engineered CRISPR-Cas Epigenetic Editors Enable Durable and Efficient Gene Silencing in Vivo
| PRJNA1273918 | ENA
Project description:mRNA-Engineered CRISPR-Cas Epigenetic Editors Enable Durable and Efficient Gene Silencing in Vivo [WGBS]
| PRJNA1273163 | ENA
Project description:mRNA-Engineered CRISPR-Cas Epigenetic Editors Enable Durable and Efficient Gene Silencing in Vivo [RNA-seq]
| PRJNA1273879 | ENA
Project description:Development of CRISPR-based epigenome editing tools is important for the study and engineering of biological behavior. Here, we describe the design of a reporter system for quantifying the ability of CRISPR epigenome editors to produce a stable gene repression. We characterize the dynamics of durable gene silencing and reactivation, as well as the induced epigenetic changes of this system. We report the creation of single-protein CRISPR constructs bearing combinations of three epigenetic editing domains, termed KAL, that can stably repress the gene expression. This system should allow for the development of novel epigenome editing tools which will be useful in a wide array of biological research and engineering applications.
| S-EPMC10521745 | biostudies-literature
Project description:Harnessing the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system for genome editing in eukaryotes has revolutionized basic biomedical research and translational sciences. The ability to create targeted alterations of the genome through this easy to design system has presented unprecedented opportunities to treat inherited disorders and other diseases such as cancer through gene therapy. A major hurdle is the lack of an efficient and safe in vivo delivery system, limiting most of the current gene therapy efforts to ex vivo editing of extracted cells. Here we discuss the unique features of adenoviral vectors that enable tissue specific and efficient delivery of the CRISPR-Cas machinery for in vivo genome editing.
| S-EPMC8091654 | biostudies-literature
Project description:Prime editor (PE) is a precise genome-editing tool capable of all possible base conversions, as well as insertions and deletions without DSBs or donor DNA. The efficient delivery of PE in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), editing efficiency at different gene loci varies among split sites, and efficient split sites within Cas9 nickase are limited. In this study, by screening multiple split sites, we demonstrated a series of efficient split site when delivering PE by dual-AAV. Additionally, we utilized a feature reported by others recently that RNase could be detached from the Cas9n and designed split sites in the first half of Cas9n. To test the editing efficiency in vivo, a novel dual-AAV split-ePE3 was packaged in AAV9 and delivered via tail vein injection in mice, achieving 24.4% precise genome editing 3 weeks post-injection. Our findings establish an alternative split-PE architecture that could achieve robust gene editing efficiency, facilitating the potential utility both in model organisms and as a therapeutic modality.
2024-12-01 | GSE270813 | GEO
Project description:Prime editor (PE) is a precise genome-editing tool capable of all possible base conversions, as well as insertions and deletions without DSBs or donor DNA. The efficient delivery of PE in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), editing efficiency at different gene loci varies among split sites, and efficient split sites within Cas9 nickase are limited. In this study, by screening multiple split sites, we demonstrated a series of efficient split site when delivering PE by dual-AAV. Additionally, we utilized a feature reported by others recently that RNase could be detached from the Cas9n and designed split sites in the first half of Cas9n. To test the editing efficiency in vivo, a novel dual-AAV split-ePE3 was packaged in AAV9 and delivered via tail vein injection in mice, achieving 24.4% precise genome editing 3 weeks post-injection. Our findings establish an alternative split-PE architecture that could achieve robust gene editing efficiency, facilitating the potential utility both in model organisms and as a therapeutic modality.
2025-03-15 | GSE291960 | GEO
Project description:Prime editor (PE) is a precise genome-editing tool capable of all possible base conversions, as well as insertions and deletions without DSBs or donor DNA. The efficient delivery of PE in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), editing efficiency at different gene loci varies among split sites, and efficient split sites within Cas9 nickase are limited. In this study, by screening multiple split sites, we demonstrated a series of efficient split site when delivering PE by dual-AAV. Additionally, we utilized a feature reported by others recently that RNase could be detached from the Cas9n and designed split sites in the first half of Cas9n. To test the editing efficiency in vivo, a novel dual-AAV split-ePE3 was packaged in AAV9 and delivered via tail vein injection in mice, achieving 24.4% precise genome editing 3 weeks post-injection. Our findings establish an alternative split-PE architecture that could achieve robust gene editing efficiency, facilitating the potential utility both in model organisms and as a therapeutic modality.
2024-12-01 | GSE270854 | GEO