PUBLICATION
Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions
- Authors
- Zhang, W., Petri, K., Ma, J., Lee, H., Tsai, C.L., Joung, J.K., Yeh, J.J.
- ID
- ZDB-PUB-240608-4
- Date
- 2024
- Source
- eLIFE 12: (Journal)
- Registered Authors
- Yeh, Jing-Ruey (Joanna)
- Keywords
- RNA structure, gene editing, genetics, genomics, protein delivery, zebrafish
- MeSH Terms
-
- Animals
- CRISPR-Associated Protein 9/genetics
- CRISPR-Associated Protein 9/metabolism
- CRISPR-Cas Systems*
- Embryo, Nonmammalian/metabolism
- Gene Editing*/methods
- RNA Folding
- RNA, Guide, CRISPR-Cas Systems*/genetics
- RNA, Guide, CRISPR-Cas Systems*/metabolism
- Zebrafish*/genetics
- PubMed
- 38847802 Full text @ Elife
Citation
Zhang, W., Petri, K., Ma, J., Lee, H., Tsai, C.L., Joung, J.K., Yeh, J.J. (2024) Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions. eLIFE. 12:.
Abstract
CRISPR prime editing (PE) requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (pegRNA), an extended version of a standard guide RNA (gRNA) that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here, we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as sixfold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping