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

• Published in: Pharmaceutics Vol. 15; no. 4; p. 1311
• Main Authors: Lee, Mei-Hwa, Thomas, James L., Lin, Chien-Yu, Li, Yi-Chen Ethan, Lin, Hung-Yin
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.04.2023; MDPI
• Subjects: Adsorption; Analysis; Chemical properties; Communication; CRISPR; CRISPR/dCas9a; Diabetes; gene activation; Gene expression; Glucose; Insulin; Magnetic fields; Methods; molecular imprinting; Nanocomposites; Nanoparticles; Peptides; Physiological aspects; Polymers; Proteins; Transfection; United States; United States > US; insulin; gene activation; molecular imprinting; gene expression; CRISPR/dCas9a
• ISSN: 1999-4923; 1999-4923
• DOI: 10.3390/pharmaceutics15041311

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.