Ontology highlight
ABSTRACT:
PROVIDER: PRJNA906920 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22475500_1.fastq.gz | Fastqsanger.gz | |||
| SRR22475500_2.fastq.gz | Fastqsanger.gz | |||
| SRR22475501_1.fastq.gz | Fastqsanger.gz | |||
| SRR22475501_2.fastq.gz | Fastqsanger.gz | |||
| SRR22461066_1.fastq.gz | Fastqsanger.gz |
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Project description:The goal of these experiments were to test the on-target and target-adjacent editing efficiencies of different single-nucleobase editing systems. Previous studies have shown that tethering DNA mutating enzymes to Cas9-nickase-UGI complexes results in editing of chromosomal DNA. However, these editing events encompass undesirable target-adjacent nucleobase edits. Here, we characterize a novel approach that reduces the frequency of target-adjacent editing while maintaining a high level of on-target editing.
2020-02-23 | E-MTAB-8742 | biostudies-arrayexpress
Project description:Prime editing allows precise modification of genomes. 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 (SSB). Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4, PE5), edit types (substitutions, insertions, deletions), guide RNA designs (pegRNAs, epegRNAs), endogenous loci, and cell types, but has no consistent effect on genome editing approaches that rely on standard, unextended guide RNAs. La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts and protects those transcripts from cellular exonucleases. We found that La stabilizes (e)pegRNAs, with particular effect on their 3' extensions, where edits are encoded. Guided by these insights, we developed a prime editing protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed (e)pegRNAs and synthetic pegRNAs optimized for La binding. 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.
2024-02-04 | GSE255003 | GEO
Project description:Prime editor (PE) has wide application prospects in disease treatment due to its diversity of editing outcomes. However, the editing efficiency of PE still needs to be further improved for therapeutic applications. Here, we increase the probability of hybridization of the pegRNA primer binding site (PBS) to single-stranded DNA flap by adding additional PBS sequence and reverse transcription template (RTT) to the loop region of pegRNA. The selection of loop and the design of loop length are optimized. The resulting modified loop2 epegRNA (ML-epegRNA) showed higher prime editing efficiency than that of the epegRNA in HEK293T cells. In addition, we used ML-epegRNA for PE editing of multiple editing types in multiple cell lines, and the results showed a general improvement in editing efficiency. Overall, the ML-epegRNA expands the capabilities of genome editing tools.
2025-11-17 | GSE274737 | 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.
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Project description:Prime editors (PEs) can mediate versatile genome editing but their efficiency remains low. Here, we developed spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE’s single-base editing efficiency or apegRNA by altering the pegRNA secondary structure to increase PE’s indel-editing efficiency . When used in PE3 and PE5, the efficiencies of sPE3, aPE3, sPE5 and aPE5 were all enhanced significantly.
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Project description:Prime editing is a novel genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct a wide variety of genetic defects.
We succeeded in efficient prime editing and functional recovery of disease-causing mutations in patient-derived liver and intestinal stem cell organoids. Whole genome sequencing of did not detect off-target mutations or a mutational signature induced by prime editing.
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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:Enhanced prime editing systems by manipulating cellular determinants of editing outcomes
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