Upgraded circular single-stranded DNA regulators for multiple-input multiple-output gene circuits in mammalian cells

Synthetic gene networks hold promise for genetic diagnostics and gene therapy but face limitations due to insufficient molecular tools. Gene-encoded circular single-stranded DNA (Css DNA) has been developed as a switchable vector to enrich regulatory components beyond protein/RNA-based systems in mammalian cells. However, the previous Css DNA regulator suffered from constrained regulatory sequence flexibility, disability of multiple-input multiple-output (MIMO) signals, and lack of endogenous orthogonal regulation. Here, we address these challenges by engineering a “bridge” design into the Css DNA regulator. These bridges function as sequence-programmable switches to control gene expression, responding to endogenous molecular signals (such as ATP, APE1, and RNase H) and enabling trans-regulation within or between Css DNAs. We exploit the orthogonality of Css DNA regulator to construct the three-input three-output genetic circuits. The upgraded Css DNA–based regulatory strategy represents a versatile and powerful platform for gene regulation and provides a promising avenue for the development of synthetic gene networks.