Mutation-specific reporter for optimization and enrichment of prime editing.

Autor: 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.
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Zdroj: Nature Communications; 10/23/2022, Vol. 13 Issue 1, p1-10, 10p
Abstrakt: 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. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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