Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design.
Proc Natl Acad Sci U S A
; 116(18): 8693-8698, 2019 04 30.
Article
em En
| MEDLINE
| ID: mdl-30988204
ABSTRACT
While CRISPR/Cas9 is a powerful tool in genome engineering, the on-target activity and off-target effects of the system widely vary because of the differences in guide RNA (gRNA) sequences and genomic environments. Traditional approaches rely on separate models and parameters to treat on- and off-target cleavage activities. Here, we demonstrate that a free-energy scheme dominates the Cas9 editing efficacy and delineate a method that simultaneously considers on-target activities and off-target effects. While data-driven machine-learning approaches learn rules to model particular datasets, they may not be as transferrable to new systems or capable of producing new mechanistic insights as principled physical approaches. By integrating the energetics of R-loop formation under Cas9 binding, the effect of the protospacer adjacent motif sequence, and the folding stability of the whole single guide RNA, we devised a unified, physical model that can apply to any cleavage-activity dataset. This unified framework improves predictions for both on-target activities and off-target efficiencies of spCas9 and may be readily transferred to other systems with different guide RNAs or Cas9 ortholog proteins.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Guia de Cinetoplastídeos
/
Sistemas CRISPR-Cas
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article