Ontology highlight
ABSTRACT:
PROVIDER: PRJNA746489 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15130700_1.fastq.gz | Fastqsanger.gz | |||
| SRR15130700_2.fastq.gz | Fastqsanger.gz | |||
| SRR15130701_1.fastq.gz | Fastqsanger.gz | |||
| SRR15130701_2.fastq.gz | Fastqsanger.gz | |||
| SRR15130702_1.fastq.gz | Fastqsanger.gz |
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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:We performed whole genome sequencing to detect possible off-target mutations induced by prime editing. Liver organoids, derived from a healthy control, were transfected with either control (GFP) plasmids or prime editing plasmids (GFP+PE2+pegRNA+nickRNA) to induce a 6-bp deletion in CTNNB1. One control and two prime-edited organoid lines were clonally expanded from single cells. High-throughput sequencing was performed on the complete genomic DNA isolated from these clonal lines, as well as the starting culture (bulk). After correction for germline mutations in the starting culture, new mutations in the control and prime-edited lines were compared. The same approach was followed in small intestinal organoids, derived from a patient with disease-causing 3-bp deletion in DGAT1. In these small intestinal organoids, prime editing was used to insert the 3 missing nucleotides. Two corrected clones were compared to one control clone.
| EGAD00001006352 | EGA
Project description:Prime editing is a powerful means of introducing precise changes to specific locations in mammalian genomes. However, the widely varying efficiency of prime editing across target sites of interest has limited its adoption in the context of both basic research and clinical settings. Here, we set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing efficiency. Utilizing a newly developed and highly sensitive method for mapping the genomic locations of a randomly integrated “sensor”, we identify specific epigenetic features that strongly correlate with the highly variable efficiency of prime editing across different genomic locations. Next, to assess the interaction of trans-acting factors with the cis-chromatin environment, we develop and apply a pooled genetic screening approach with which the impact of knocking down various DNA repair factors on prime editing efficiency can be stratified by cis-chromatin context. Finally, we demonstrate that we can dramatically modulate the efficiency of prime editing through epigenome editing, i.e. enhancing (or restricting) local chromatin accessibility in order to increase (or decrease) the efficiency of prime editing at a target site. Looking forward, we envision that the insights and tools described here will broaden the range of both basic research and therapeutic contexts in which prime editing is useful.
2023-04-12 | GSE228462 | GEO
Project description:Prime editing is a powerful means of introducing precise changes to specific locations in mammalian genomes. However, the widely varying efficiency of prime editing across target sites of interest has limited its adoption in the context of both basic research and clinical settings. Here, we set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing efficiency. Utilizing a newly developed and highly sensitive method for mapping the genomic locations of a randomly integrated “sensor”, we identify specific epigenetic features that strongly correlate with the highly variable efficiency of prime editing across different genomic locations. Next, to assess the interaction of trans-acting factors with the cis-chromatin environment, we develop and apply a pooled genetic screening approach with which the impact of knocking down various DNA repair factors on prime editing efficiency can be stratified by cis-chromatin context. Finally, we demonstrate that we can dramatically modulate the efficiency of prime editing through epigenome editing, i.e. enhancing (or restricting) local chromatin accessibility in order to increase (or decrease) the efficiency of prime editing at a target site. Looking forward, we envision that the insights and tools described here will broaden the range of both basic research and therapeutic contexts in which prime editing is useful.
2023-04-12 | GSE228463 | GEO
Project description:Prime editing is a powerful means of introducing precise changes to specific locations in mammalian genomes. However, the widely varying efficiency of prime editing across target sites of interest has limited its adoption in the context of both basic research and clinical settings. Here, we set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing efficiency. Utilizing a newly developed and highly sensitive method for mapping the genomic locations of a randomly integrated “sensor”, we identify specific epigenetic features that strongly correlate with the highly variable efficiency of prime editing across different genomic locations. Next, to assess the interaction of trans-acting factors with the cis-chromatin environment, we develop and apply a pooled genetic screening approach with which the impact of knocking down various DNA repair factors on prime editing efficiency can be stratified by cis-chromatin context. Finally, we demonstrate that we can dramatically modulate the efficiency of prime editing through epigenome editing, i.e. enhancing (or restricting) local chromatin accessibility in order to increase (or decrease) the efficiency of prime editing at a target site. Looking forward, we envision that the insights and tools described here will broaden the range of both basic research and therapeutic contexts in which prime editing is useful.
2023-04-12 | GSE228464 | GEO
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:Purpose: The goals of this study are to introduce a new genome editing tool, which has the higher editing scope than the original genome editing tools. Methods: First, we transfected PE2 (the original prime editing tool, prime editor2), PE3 (the original prime editing tool, prime editor3) and HOPE (the new tool we developed in this study) vectors into human cells, respectively. Then, we harvested the genomic DNA form the transfected cells and amplified the specified amplicons. Finally, we used targeted amplicon sequencing approach to compare the editing efficiency and presion of the new tool with the original reported tools. Results: Our new genome editing tool improves the editing efficiency of prime editing without increasing the risk of undesired indels formation. Conclusions: We deleveped a new genome editing tool to increase the likelihood of successful gene engineering.
2021-04-10 | GSE171470 | GEO