Activation of Insulin Gene Expression via Transfection of a CRISPR/dCas9a System Using Magnetic Peptide-Imprinted Nanoparticles

Mei-Hwa Lee; James L Thomas; Chien-Yu Lin; Yi-Chen Ethan Li; Hung-Yin Lin

doi:10.3390/pharmaceutics15041311

Activation of Insulin Gene Expression via Transfection of a CRISPR/dCas9a System Using Magnetic Peptide-Imprinted Nanoparticles

Pharmaceutics. 2023 Apr 21;15(4):1311. doi: 10.3390/pharmaceutics15041311.

Authors

Mei-Hwa Lee¹, James L Thomas², Chien-Yu Lin³, Yi-Chen Ethan Li⁴, Hung-Yin Lin³

Affiliations

¹ Department of Materials Science and Engineering, I-Shou University, Kaohsiung 84001, Taiwan.
² Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM 87131, USA.
³ Department of Chemical and Materials Engineering, National University of Kaohsiung, Kaohsiung 81148, Taiwan.
⁴ Department of Chemical Engineering, Feng Chia University, Taichung 40724, Taiwan.

Abstract

A CRISPRa transcription activation system was used to upregulate insulin expression in HEK293T cells. To increase the delivery of the targeted CRISPR/dCas9a, magnetic chitosan nanoparticles, imprinted with a peptide from the Cas9 protein, were developed, characterized, and then bound to dCas9a that was complexed with a guide RNA (gRNA). The adsorption of dCas9 proteins conjugated with activators (SunTag, VPR, and p300) to the nanoparticles was monitored using both ELISA kits and Cas9 staining. Finally, the nanoparticles were used to deliver dCas9a that was complexed with a synthetic gRNA into HEK293T cells to activate their insulin gene expression. Delivery and gene expression were examined using quantitative real-time polymerase chain reaction (qRT-PCR) and staining of insulin. Finally, the long-term release of insulin and the cellular pathway related to stimulation by glucose were also investigated.

Keywords: CRISPR/dCas9a; gene activation; gene expression; insulin; molecular imprinting.

Abstract

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