Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions.
Elife
; 122024 Jun 07.
Article
en En
| MEDLINE
| ID: mdl-38847802
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.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Pez Cebra
/
Sistemas CRISPR-Cas
/
Edición Génica
/
ARN Guía de Sistemas CRISPR-Cas
Límite:
Animals
Idioma:
En
Año:
2024
Tipo del documento:
Article