Mus musculus strain:C57BL/6 x DBA2
Ontology highlight
ABSTRACT:
PROVIDER: PRJDB19128 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| DRR615897.fastq.gz | Fastqsanger.gz | |||
| DRR615897_1.fastq.gz | Fastqsanger.gz | |||
| DRR615897_2.fastq.gz | Fastqsanger.gz | |||
| DRR615898.fastq.gz | Fastqsanger.gz | |||
| DRR615898_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 9 |
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Project description:Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3′ ends of CRISPR–Cas guide RNAs. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3′ ends of RNA polymerase III transcripts. We found that La functionally interacts with the 3′ ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.
2024-01-22 | GSE253424 | GEO
Project description:We evaluate CRISPR-based prime editing for application in organoids. First we model mutations in TP53 in intestinal and hepatocyte oganoids and determine the efficiency and accuracy of mutation induction on multiple targets. Then, to evaluate potential clinical applicability of prime editing we repair mutations in the CFTR channel that cause cystic fibrosis in intestinal organoids. First we repair the CFTR-F508del mutation which is the most common mutation in cystic fibrosis. Then we compare adenine base editing to prime editing by repairing the CFTR-R785* mutation using both strategies.
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Project description:Genome-wide Profiling of off-targets from Prime editor in vitro and in vivo using PE-Tag
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Project description:Prime editing is a versatile genome-editing technique that shows great promise for the generation and repair of patient mutations. However, some genomic sites are difficult to edit and optimal design of prime-editing tools remains elusive. Here we present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) combined with any prime editor variant. We apply fluoPEER to instruct correction of pathogenic variants in patient cells and find that plasmid-editing enriches for genomic editing up to 3-fold compared to conventional enrichment strategies. DNA repair and cell cycle-related genes are enriched in the transcriptome of edited cells. Stalling cells in the G1/S boundary increases prime editing efficiency up to 30%. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.
2022-02-03 | GSE195977 | 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:Translational research is commonly performed in the C57B6/J mouse strain, chosen for its genetic homogeneity and phenotypic uniformity. Here, we evaluate the suitability of the white-footed deer mouse (Peromyscus leucopus) as a model organism for aging research, offering a comparative analysis against C57B6/J and diversity outbred (DO) Mus musculus strains. Our study includes comparisons of body composition, skeletal muscle function, and cardiovascular parameters, shedding light on potential applications and limitations of P. leucopus in aging studies. Notably, P. leucopus exhibits distinct body composition characteristics, emphasizing reduced muscle force exertion and a unique metabolism, particularly in fat mass. Cardiovascular assessments showed changes in arterial stiffness, challenging conventional assumptions and highlighting the need for a nuanced interpretation of aging-related phenotypes. Our study also highlights inherent challenges associated with maintaining and phenotyping P. leucopus cohorts. Behavioral considerations, including anxiety-induced responses during handling and phenotyping assessment, pose obstacles in acquiring meaningful data. Moreover, the unique anatomy of P. leucopus necessitates careful adaptation of protocols designed for Mus musculus. While showcasing potential benefits, further extensive analyses across broader age ranges and larger cohorts are necessary to establish the reliability of P. leucopus as a robust and translatable model for aging studies.
| S-EPMC11335981 | biostudies-literature