Strategic engineering of DNA aptamers as precision modulators in CRISPR-driven oncogene control

The goal of this study was to address the limited availability of intracellular protein-binding aptamers that restrict the potential of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based signal conductor systems. We aimed to develop a programmable CRISPR-SaCas9 platform using DNA aptamers to achieve precise and dynamic gene regulation for cancer therapy.

We identified and characterized five DNA aptamers with high specificity for the SaCas9 protein. Functional assays were performed to evaluate their effects on the transcriptional regulatory activity of SaCas9. Some aptamers were engineered to associate with promoter elements, enabling transcription factor-responsive control of downstream gene expression. To test therapeutic efficacy, the system was delivered in vivo using In vivo-jetPEI reagents, and tumor progression and metastasis were monitored.

The identified aptamers exhibited distinct functionalities - some partially inhibited SaCas9 activity, while others enabled precise gene expression control in response to endogenous transcription factors. The aptamer-CRISPR complexes formed ternary assemblies with SaCas9 and single-guide RNAs, enabling logic-gated responses. In vivo , the system significantly suppressed tumor growth and metastasis.

We developed a modular CRISPR signal conductor platform that leverages SaCas9-binding DNA aptamers for transcription factor-mediated logic operations and precise gene control. This system expands the toolkit for genetic circuit design and offers a promising strategy for targeted cancer gene therapy.