Mutation-specific reporter for optimization and enrichment of prime editing

Mutation-specific reporter for optimization and enrichment of prime editing

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 r...

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Bibliographic Details
Published in:Nature communications Vol. 13; no. 1; p. 1028
Main Authors: Schene, I. F., Joore, I. P., Baijens, J. H. L., Stevelink, R., Kok, G., Shehata, S., Ilcken, E. F., Nieuwenhuis, E. C. M., Bolhuis, D. P., van Rees, R. C. M., Spelier, S. A., van der Doef, H. P. J., Beekman, J. M., Houwen, R. H. J., Nieuwenhuis, E. E. S., Fuchs, S. A.
Format: Journal Article
Language:English
Published: London Nature Publishing Group UK 01-03-2022
Nature Publishing Group
Nature Portfolio
Subjects:
13/100
45/41
49
49/23
49/31
49/91
631/1647/1511
631/208/4041/3196
631/61/201/2110
631/92/147
692/4017
Cell cycle
CRISPR-Cas Systems - genetics
DNA repair
Editing
Efficiency
Enrichment
Fluorescence
Gene Editing - methods
Genetic modification
Genome
Genome editing
Genomics
Humanities and Social Sciences
Humans
multidisciplinary
Mutation
Optimization
Repair
RNA, Guide, CRISPR-Cas Systems
Science
Science (multidisciplinary)
Stalling
Transcriptomes
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Description
Summary: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. While prime editing is a promising technique, some genomic sites remain difficult to edit. Here the authors present fluoPEER, fluorescent prime editing and enrichment reporter, to rank the efficiency of pegRNAs and prime editor variants.
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content type line 23
ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-022-28656-3