A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Effective Delivery of the CRISPR/Cas9 System Enabled by Functionalized Mesoporous Silica Nanoparticles for GFP-Tagged Paxillin Knock-In
Tekijät: Xu Xiaoyu, Koivisto Oliver, Liu Chang, Zhou Junnian, Miihkinen Mitro, Jacquemet Guillaume, Wang Daqi, Rosenholm Jessica M, Shu Yilai, Zhang Hongbo
Kustantaja: WILEY
Julkaisuvuosi: 2021
Journal: Advanced therapeutics
Tietokannassa oleva lehden nimi: ADVANCED THERAPEUTICS
Lehden akronyymi: ADV THER-GERMANY
Artikkelin numero: 2000072
Vuosikerta: 4
Numero: 1
Sivujen määrä: 12
eISSN: 2366-3987
DOI: https://doi.org/10.1002/adtp.202000072
Rinnakkaistallenteen osoite: https://research.utu.fi/converis/portal/detail/Publication/53035775
In this study, direct and effective intracellular delivery of CRISPR/Cas9 plasmids for homology-directed repair is achieved by functionalized mesoporous silica nanoparticles (MSNs). The functionalized MSNs (Cy5.5-MSNs-NLS) are synthesized by in situ labeling of a fluorescent dye (Cy5.5) and surface conjugation of nuclear localization sequence (NLS, PKKKRKV), showing a high loading efficiency (50%) toward the plasmids (PXN cutdown plasmid: GFP-Cas9-paxillin_gRNA and repair plasmid: AICSDP-1: PXN-EGFP). Subsequently, a polymeric coating of the poly(dimethyldiallylammonium chloride) (PDDA) is electrostatically deposited onto the plasmid-loaded Cy5.5-MSNs-NLS by microfluidic nanoprecipitation. The coating layer offers effective protection against the denaturation of plasmids by EcoRV restriction enzymes, and is shown to prevent premature release. Moreover, owing to the positive charge and pH-responsive disaggregation of PDDA, enhanced cellular internalization (16 h) and endosomal escape (4 h) of the nanocarrier are observed. After escape of nanocarrier system into the cytoplasm, the NLS on the surface of MSNs facilitates nuclear transport of the CRISPR/Cas9 plasmids, achieving successful GFP-tag knock-in of the PXN genomic sequence in U2OS cells. This intracellular delivery system thus offers an attractive method to overcome physiological barriers for CRISPR/Cas9 delivery, showing considerable promise for paxillin-associated focal adhesion and signaling regulator investigation.
Ladattava julkaisu This is an electronic reprint of the original article. |  |
