Smaruj, P. N.; Kelley, D. R.; Fudenberg, G.

Mammalian genomes display complex three-dimensional organization which is crucial for downstream processes like gene regulation. Local features of genome organization are largely driven by loop extrusion and manifest as boundaries, dots, and flames in genome contact maps. Still, the rational design of DNA sequences that produce desired folding patterns has not been demonstrated. Here, we present Akita Semifreddo, a framework that enables the rational in silico design of DNA sequences with programmable 3D folding outcomes. This combines a computationally efficient "half-frozen" version of the AkitaV2 genome folding model with the Ledidi sequence optimizer. We systematically demonstrate that this approach spans the full repertoire of known local folding features. We show that ~2 kb synthetic sequences can be designed to induce boundaries, dots, and flames at desired strengths, with CTCF motif configurations consistent with their known mechanistic bases. We further demonstrate that weak boundaries can be designed through transcription-associated sequence features alone, without introducing CTCF motifs, and that strong boundaries can be suppressed by introducing SINE B2 retroelement-like sequences. Collectively, these results reveal a many-to-one relationship between DNA sequence and folding outcomes and uncover the biological basis of sequence features leveraged by our model. In short, Akita Semifreddo provides a platform for dissecting the sequence grammar of three-dimensional chromatin architecture and engineering synthetic regulatory landscapes.