Ultra-high-throughput mapping of genetic design space

Massively parallel genetic screens have been used to map sequence-to-function relationships for a variety of genetic elements^1-5. However, as these approaches interrogate only short sequences, it remains challenging to perform high-throughput assays on constructs containing combinations of multiple sequence elements arranged across multi-kb length scales. Overcoming this barrier could accelerate synthetic biology; by screening diverse gene circuit designs and learning 'composition to function' mappings, genetic part composability rules could be revealed, enabling rapid identification of behaviour-optimized design variants^6,7. Here we introduce CLASSIC (combining long- and short-range sequencing to investigate genetic complexity), a genetic screening platform that combines long- and short-read next-generation sequencing (NGS) modalities to quantitatively assess pools of constructs of arbitrary length containing diverse genetic part compositions. We show that CLASSIC can measure expression profiles of over 10⁵ gene circuit designs (from 5-20 kb) in a single experiment in human cells. The resulting datasets can be used to train machine-learning models that accurately predict circuit behaviour across expansive circuit design landscapes, revealing part composability rules that govern circuit performance. Our study shows that, by expanding the throughput of each design-build-test-learn cycle, CLASSIC enhances the pace and scale of synthetic biology and establishes an experimental basis for data-driven design of complex genetic systems.