A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.
J Biol Chem
; 300(7): 107439, 2024 Jul.
Article
em En
| MEDLINE
| ID: mdl-38838774
ABSTRACT
The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pareamento Incorreto de Bases
/
Sistemas CRISPR-Cas
/
Proteína 9 Associada à CRISPR
Limite:
Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Índia