Lessons from Enzyme Kinetics Reveal Specificity Principles for RNA-Guided Nucleases in RNA Interference and CRISPR-Based Genome Editing.
Cell Syst
; 4(1): 21-29, 2017 01 25.
Article
em En
| MEDLINE
| ID: mdl-28125791
ABSTRACT
RNA-guided nucleases (RGNs) provide sequence-specific gene regulation through base-pairing interactions between a small RNA guide and target RNA or DNA. RGN systems, which include CRISPR-Cas9 and RNA interference (RNAi), hold tremendous promise as programmable tools for engineering and therapeutic purposes. However, pervasive targeting of sequences that closely resemble the intended target has remained a major challenge, limiting the reliability and interpretation of RGN activity and the range of possible applications. Efforts to reduce off-target activity and enhance RGN specificity have led to a collection of empirically derived rules, which often paradoxically include decreased binding affinity of the RNA-guided nuclease to its target. We consider the kinetics of these reactions and show that basic kinetic properties can explain the specificities observed in the literature and the changes in these specificities in engineered systems. The kinetic models described provide a foundation for understanding RGN targeting and a necessary conceptual framework for their rational engineering.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Ribonucleases
/
RNA Guia de Cinetoplastídeos
/
Interferência de RNA
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Cell Syst
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Estados Unidos