CKM and TERT dual promoters drive CRISPR–dCas9 to specifically inhibit the malignant behavior of osteosarcoma cells

Abstract

Improvements in treatment and chemotherapy have increased the survival rate of osteosarcoma, but overall efficacy remains low, highlighting the need for new gene therapy methods. Clustered regularly interspaced short palindromic repeats–deactivated Cas9 (CRISPR–dCas9) technology offers a promising strategy, but targeting osteosarcoma cells precisely is a challenge. We designed a system to achieve specific expression of CRISPR–dCas9–KRAB in osteosarcoma cells by using the creatine kinase muscle (CKM) promoter to drive dCas9–KRAB and the telomerase reverse transcriptase (TERT) promoter to drive single guide (sg)RNA expression. We inhibited the MDM2 proto-oncogene using this system in vitro, which efficiently inhibited the malignant behavior of osteosarcoma cells and induced apoptosis without affecting normal cells. In vivo experiments demonstrated that this system effectively inhibited the growth of subcutaneously transplanted tumors in nude mice. These findings provide a new method for precise identification and intervention of osteosarcoma with significant implications for the development of gene therapy methods for other cancers. Future research should focus on optimizing this system for clinical translation.

Functional identification of PGM1 in the regulating development and depositing of inosine monophosphate specific for myoblasts

“…Meanwhile, an opposite trend was observed in the interference group compared with the NC-siRNA group ( Figures 2K – P ). The differentiation and development of myoblasts into multinucleated myotubes is controlled at multiple levels by a number of key transcription and regulatory factors ( 24 ). The CKM and myogenesis regulatory factors ( MYFs ) family of genes play important regulatory roles in initiating and maintaining the process of skeletal myoblast differentiation and are considered to be hallmark factors that promote myoblast differentiation ( 25 , 26 ).…”

Gene Therapy in Pediatric Orthopedics

“…These researchers fused the dCAS9 protein with the KRAB fusion protein, a transcriptional repressor domain that can silence gene expression. The CKM and TERT promoters drive the expression of dCas9-KRAB and the sgRNA in malignant OS cells, explicitly targeting the MDM2 proto-oncogene [31]. The study found that the CRISPR-dCAS9-KRAB system could specifically target and silence the MDM2 gene.…”

“…Another gene therapy approach for treating OS is to target tumor-associated protooncogenes and silence their expression to inhibit tumor growth [31]. Proto-oncogenes that can become abnormally activated in OS include MDM2, SAS and c-myc [31].…”

“…Another gene therapy approach for treating OS is to target tumor-associated protooncogenes and silence their expression to inhibit tumor growth [31]. Proto-oncogenes that can become abnormally activated in OS include MDM2, SAS and c-myc [31]. Hu et al used a CRISPR-dCAS9-KRAB system with CKM and TERT promoters to target the MDM2 proto-oncogene and inhibit its expression in human OS cells [31].…”

“…Proto-oncogenes that can become abnormally activated in OS include MDM2, SAS and c-myc [31]. Hu et al used a CRISPR-dCAS9-KRAB system with CKM and TERT promoters to target the MDM2 proto-oncogene and inhibit its expression in human OS cells [31]. CRISPR-dCAS9 can bind to a specific location guided by sgRNA and act as a scaffold to recruit effector molecules.…”