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ABSTRACT: High-throughput evaluation of prime editor activity

PROVIDER: PRJNA624815 | ENA |

REPOSITORIES: ENA

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Dataset's files

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			Action	DRS
	SRR11529289_1.fastq.gz	Fastqsanger.gz
	SRR11529289_2.fastq.gz	Fastqsanger.gz
	SRR12334115_1.fastq.gz	Fastqsanger.gz
	SRR12334115_2.fastq.gz	Fastqsanger.gz
	SRR12334116_1.fastq.gz	Fastqsanger.gz

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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.

2024-01-22 | GSE253424 | GEO

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

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

| PRJNA734350 | ENA

Project description:Deep sequencing of prime editor target sequences

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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.

2022-02-03 | GSE178112 | GEO

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

2022-02-03 | GSE178111 | GEO

Massively parallel quantification of the regulatory effects of non-coding genetic variation [STARR-seq]

Project description:We report a novel high-throughput method to empirically quantify individual-specific regulatory element activity at the population scale. The approach combines targeted DNA capture with a high-throughput reporter-gene expression assay. As demonstration, we have measured the activity of more than 100 putative regulatory elements from 95 individuals in a single experiment. We found that, in agreement with previous reports, most genetic variants have weak effects on distal regulatory element activity. Because haplotypes are typically maintained within but not between assayed regulatory elements, the approach can be used to identify likely causal regulatory haplotypes that contribute to human phenotypes. Finally, we demonstrate the utility of the method to functionally fine map causal regulatory variants in regions of high linkage disequilibrium identified by expression quantitative trait loci (eQTL) analyses. 104 candidate regulatory elements from 95 individuals were resequenced using Illumina custom amplicon sequencing. We then cloned the resulting DNA fragments into a massively parallel reporter assay to quantify allele-specific regulatory activity from that population. SNP-fdr.txt contains output of significance evaluation haplotype.fasta.gz contains the reference used to generate alignment files

2015-06-15 | E-GEOD-68331 | biostudies-arrayexpress

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