Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA811252 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18290830_1.fastq.gz | Fastqsanger.gz | |||
| SRR18290830_2.fastq.gz | Fastqsanger.gz | |||
| SRR18290831_1.fastq.gz | Fastqsanger.gz | |||
| SRR18290831_2.fastq.gz | Fastqsanger.gz | |||
| SRR18290832_1.fastq.gz | Fastqsanger.gz |
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Project description:Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.
2022-02-03 | GSE178112 | GEO
Project description:Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.
2022-02-03 | GSE178111 | GEO
Project description:Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of genetic defects1. However, correction of a disease phenotype in vivo in somatic tissues has not been achieved yet. Here, we establish proof-of-concept for in vivo prime editing, that resulted in rescue of a metabolic liver disease. We first develop a size-reduced prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (SpCas9-PERnH), and a linker- and NLS-optimized intein-split PE construct (SpCas9-PE p.1153) for delivery by adeno-associated viruses (AAV). Systemic dual AAV-mediated delivery of this variant in neonatal mice enables installation of a transversion mutation at the Dnmt1 locus with 15% efficiency on average. Next, we targeted the disease-causing mutation in the phenylalanine hydroxylase (Pah)enu2 mouse model for phenylketonuria (PKU). Correction rates of 1.5% using the dual AAV approach could be increased to up to 14% by delivery of full-length SpCas9-PE via adenoviral vector 5 (AdV5), leading to full restoration of physiological blood phenylalanine (L-Phe) levels below 120 µmol/L. Our study demonstrates in vivo prime editing in the liver at two independent loci, emphasizing the potential of PEs for future therapeutic applications.
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Project description:Repairing pathogenetic mutations using engineered Prime Editor in vivo
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Project description:We developed CROss-seq, a versatile, efficient and cost-effective method for genome-wide off-target profiling in multiple genome editing systems. Purpose: CRISPR-mediated genome editors have been widely deployed in basic research and therapeutic applications, yet the targeting specificity of these editors is still the major concern. Method to examine the genome-wide off-target effects of these editors remains lagging behind. Methods: in vivo CROss-seq, the transfected cells are labeled with N3-kethoxal for 45 minutes; in vitro CROss-seq, the genomic DNA are incubated with N3-kethoxal for 2 hours. The treated gDNA are processed with biotin click reaction, fragmentation, adapter ligation, and pull-down steps. The purified libraries were sequenced on Illumina HiSeq X Ten platform to generate paired-end reads. Results: Through CROss-seq we successfully both in vivo and in vitro to evaluate the off-target effects of CRISPR-Cas9 nuclease, base editors and prime editor. The in vivo assay was more specific while in vitro assay was more sensitive, highlighting the necessity of coupling in vivo and in vitro assays to achieve a comprehensive and precise assessment of the off-target effects. CROss-seq also revealed chromatin accessibility plays an important role in the off-target activity of the CRISPR-based editing. In addition, we identified limited genome-wide off-target sites of BE and PE in vivo, indicating that both editors were highly specific to target site.
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Project description:Nucleotide repeat expansion disorders, a group of genetic diseases characterized by the expansion of specific DNA sequences, pose significant challenges to treatment and therapy development. Here, we present a precise and programmable method called prime editor–mediated correction of nucleotide repeat expansion (PE-CORE) for correcting pathogenic nucleotide repeat expansion. PE-CORE leverages a prime editor and paired pegRNAs to achieve targeted correction of repeat sequences. We demonstrate the effectiveness of PE-CORE in HEK293T cells and patient-derived induced pluripotent stem cells (iPSCs). Specifically, we focus on spinal and bulbar muscular atrophy and spinocerebellar ataxia type, two diseases associated with nucleotide repeat expansion. Our results demonstrate the successful correction of pathogenic expansions in iPSCs and subsequent differentiation into motor neurons. Specifically, we detect distinct downshifts in the size of both the mRNA and protein, confirming the functional correction of the iPSC-derived motor neurons. These findings highlight PE-CORE as a precision tool for addressing the intricate challenges of nucleotide repeat expansion disorders, paving the way for targeted therapies and potential clinical applications.
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2024-04-22 | GSE247589 | 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
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.
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Project description:The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.
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