Programmable trans-splicing riboregulators for complex cellular logic computation

Abstract

Synthetic genetic circuits program the cellular input–output relationships to execute customized functions. However, efforts to scale up these circuits have been hampered by the limited number of reliable regulatory mechanisms with high programmability, performance, predictability and orthogonality. Here we report a class of split-intron-enabled trans-splicing riboregulators (SENTRs) based on de novo designed external guide sequences. SENTR libraries provide low leakage expression, wide dynamic range, high predictability with machine learning and low crosstalk at multiple component levels. SENTRs can sense RNA targets, process signals by logic computation and transduce them into various outputs, either mRNAs or noncoding RNAs. We subsequently demonstrate that digital logic operation with up to six inputs can be implemented using multiple orthogonal SENTRs to regulate a single gene simultaneously and coupling SENTRs with split intein-mediated protein trans-splicing. SENTR represents a powerful and versatile regulatory tool at the post-transcriptional level in Escherichia coli, suggesting broad biotechnological applications. Scaling up genetic circuits is a longstanding challenge in synthetic biology. Here Gao et al. engineer split introns for programmable RNA-level gene regulation and integrate them with protein splicing for single-layer multi-input logic computation.