[The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory].
Mol Biol (Mosk)
; 58(1): 157-159, 2024.
Article
en Ru
| MEDLINE
| ID: mdl-38943587
ABSTRACT
Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1-V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Streptococcus pyogenes
/
Sistemas CRISPR-Cas
/
Edición Génica
/
Proteína 9 Asociada a CRISPR
Límite:
Humans
Idioma:
Ru
Revista:
Mol Biol (Mosk)
Año:
2024
Tipo del documento:
Article