RESUMEN
Gene therapy is expected to be utilized for the treatment of various diseases. However, the spatiotemporal resolution of current gene therapy technology is not high enough. In this study, we generated a new technology for spatiotemporally controllable gene therapy. We introduced optogenetic and CRISPR/Cas9 techniques into a recombinant adenovirus (Ad) vector, which is widely used in clinical trials and exhibits high gene transfer efficiency, to generate an illumination-dependent spatiotemporally controllable gene regulation system (designated the Opt/Cas-Ad system). We generated an Opt/Cas-Ad system that could regulate a potential tumor suppressor gene, and we examined the effectiveness of this system in cancer treatment using a xenograft tumor model. With the Opt/Cas-Ad system, highly selective tumor treatment could be performed by illuminating the tumor. In addition, Opt/Cas-Ad system-mediated tumor treatment could be stopped simply by turning off the light. We believe that our Opt/Cas-Ad system can enhance both the safety and effectiveness of gene therapy.
Asunto(s)
Adenoviridae/genética , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/efectos de la radiación , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/efectos de la radiación , Terapia Genética/métodos , Proteínas Adaptadoras Transductoras de Señales , Animales , Línea Celular Tumoral , Quimiocinas , Endonucleasas/genética , Endonucleasas/efectos de la radiación , Femenino , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Genes Supresores de Tumor/efectos de la radiación , Vectores Genéticos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Luz , Ratones Endogámicos BALB C , ARN Guía de Kinetoplastida/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.