Reconstructing sequence-grammar trajectories enables interpretable and tunable cis- regulatory element design

Designing synthetic cis-regulatory elements (CREs) with cell-type-specific activity remains challenging, and optimization is usually treated as a black box, obscuring how regulatory grammar emerges and why design trajectories fail. Here, we present GO-CRE (Guided Optimization of Cis-Regulatory Elements), which combines efficient sequence generation, predictor-guided reinforcement learning, and trajectory-level interpretation. GO-CRE reconstructs iterative sequence changes in a shared sequence-grammar landscape and identifies coordinated update programs corresponding to search, commitment, and optimization. Productive trajectories in HepG2 and K562 progressively acquired cell-type-associated grammar, whereas SK-N-SH trajectories remained confined to local basins. In HepG2, trajectory analysis revealed a low-complexity polyG trap; introducing a polyG penalty redirected optimization toward HNF/FOXA-associated features. Final designs retained sequence diversity while converging on cell-type-associated motif patterns. Lentiviral MPRA validated cell-type-specific activity in K562 and HepG2 and showed higher average activity of HepG2 designs than endogenous CREs. Together, these findings establish sequence-grammar trajectory reconstruction as a basis for interpretable and tunable synthetic CRE design.