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Precise editing of pathogenic nucleotide repeat expansion in iPSCs without inducing DNA double-strand breaks using a prime editor [RNA-Seq]

ABSTRACT: Precise editing of pathogenic nucleotide repeat expansion in iPSCs without inducing DNA double-strand breaks using a prime editor [RNA-Seq]

PROVIDER: PRJNA1041748 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR26860514_1.fastq.gz	Fastqsanger.gz
	SRR26860514_2.fastq.gz	Fastqsanger.gz
	SRR26860515_1.fastq.gz	Fastqsanger.gz
	SRR26860515_2.fastq.gz	Fastqsanger.gz
	SRR26860516_1.fastq.gz	Fastqsanger.gz

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Precise editing of pathogenic nucleotide repeat expansion in iPSCs without inducing DNA double-strand breaks using a prime editor [RNA-Seq]

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.

2024-06-12 | GSE248085 | GEO

No observable guide-RNA-independent off-target mutation induced by prime editor [TargetSeq]

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.

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No observable guide-RNA-independent off-target mutation induced by prime editor [RNA-Seq]

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Easy-Prime: a machine learning–based prime editor design tool

Project description:Easy-Prime: a machine learning–based prime editor design tool

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Improving prime editing with an endogenous small RNA-binding protein

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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Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways [WES]

Project description:Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived human pluripotent stem cells (hPSCs). Unlike Cas9-mediated knock-in, cytosine base editor (CBE) and prime editor (PE) achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by CBE or PE additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of dominant negative p53 (p53DD) and three UNG inhibitor (UGI), engineered to specifically diminish base excision repair (BER), improved CBE efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional PE system also significantly enhanced PE efficiency in hPSCs. Thus, combined inhibition of the unique cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

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Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways [RNA-seq]

2024-04-22 | GSE247589 | GEO

Improving prime editing with an endogenous small RNA-binding protein

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.

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No observable guide-RNA-independent off-target mutation induced by prime editor

Project description:No observable guide-RNA-independent off-target mutation induced by prime editor

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No observable guide-RNA-independent off-target mutation induced by prime editor [TargetSeq]

Project description:No observable guide-RNA-independent off-target mutation induced by prime editor [TargetSeq]

| PRJNA737412 | ENA