Cationic lipopolymeric nanoplexes containing the CRISPR/Cas9 ribonucleoprotein for genome surgery.
J Mater Chem B
; 10(37): 7634-7649, 2022 09 28.
Article
em En
| MEDLINE
| ID: mdl-35946380
ABSTRACT
sgRNA/Cas9 ribonucleoproteins (RNPs) provide a site-specific robust gene-editing approach avoiding the mutagenesis and unwanted off-target effects. However, the high molecular weight (â¼165 kDa), hydrophilicity and net supranegative charge (â¼-20 mV) hinder the intracellular delivery of these RNPs. In the present study, we have prepared cationic RNPs lipopolymeric nanoplexes that showed a size of 117.3 ± 7.64 nm with +6.17 ± 1.04 mV zeta potential and >90% entrapment efficiency of RNPs. Further, these RNPs lipopolymeric nanoplexes showed good complexation efficiency and were found to be stable for 12 h with fetal bovine serum. These RNPs lipopolymeric nanoplexes did not induce any significant cytotoxicity in HEK293T cells, and were efficiently uptaken via a clathrin-mediated pathway with optimal transfection efficiency and nuclear localization after 48 h. Further, HEK293T cells having the mGFP insert were used as a cell line model for gene editing, wherein the loss of the mGFP signal was observed as a function of gene editing after transfection with mGFP targeting RNPs lipopolymeric nanoplexes. Further, the T7 endonuclease and TIDE assay data showed a decent gene editing efficiency. Additionally, the lipopolymeric nanoplexes were able to transfect muscle cells in vivo, when injected intra-muscularly. Collectively, this study explored the potential of cationic lipopolymeric nanoplexes for delivering gene-editing endonucleases.
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Base de dados:
MEDLINE
Assunto principal:
Ribonucleoproteínas
/
Sistemas CRISPR-Cas
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article