Issue 37, 2022

Cationic lipopolymeric nanoplexes containing the CRISPR/Cas9 ribonucleoprotein for genome surgery

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 (∼165 kDa), hydrophilicity and net supranegative charge (∼−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.

Graphical abstract: Cationic lipopolymeric nanoplexes containing the CRISPR/Cas9 ribonucleoprotein for genome surgery

Supplementary files

Article information

Article type
Paper
Submitted
24 mar 2022
Accepted
19 jul 2022
First published
20 jul 2022

J. Mater. Chem. B, 2022,10, 7634-7649

Cationic lipopolymeric nanoplexes containing the CRISPR/Cas9 ribonucleoprotein for genome surgery

D. K. Sahel, M. Salman, M. Azhar, S. G. Goswami, V. Singh, M. Dalela, S. Mohanty, A. Mittal, S. Ramalingam and D. Chitkara, J. Mater. Chem. B, 2022, 10, 7634 DOI: 10.1039/D2TB00645F

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