✍🏼 Ronan W. O’Connell, Kshitij Rai, Trenton C. Piepergerdes, Kian D. Samra, Jack
A. Wilson, Shujian Lin, Thomas H. Zhang, Eduardo M. Ramos, Andrew Sun, Bryce
Kille, Kristen D. Curry, Jason W. Rocks, Todd J. Treangen, Pankaj Mehta, and
Caleb J. Bashor
🏠 Department of Bioengineering, Rice University, Houston, TX, USA
📑 bioRxiv (2025)
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Abtract
Massively parallel genetic screens have been used to map sequence-to-function relationships for a variety of genetic elements. However, because these approaches only interrogate short sequences, it remains challenging to perform high throughput (HT) 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 that reveal genetic part composability rules and enable rapid identification of behavior-optimized variants. Here, we introduce CLASSIC, 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 part compositions. We show that CLAS-SIC can measure expression profiles of >105 gene circuit designs (from 5-20 kb) in a single experiment in human cells. The resulting datasets can be used to train ML models that accurately predict circuit behavior across expansive circuit design landscapes, revealing part composability rules that govern circuit performance. Our work shows that by expanding the throughput of each design-build-test-learn (DBTL) cycle, CLASSIC enhances the pace and scale of synthetic biology and establishes an experimental basis for data-driven design of complex genetic systems.
How the WOLF is used in this study
The authors used the WOLF cell sorter as part of their workflow to isolate genetically edited clones after library integration and selection. After introducing genetic variants into cells and selecting for integration with antibiotic resistance (e.g., hygro‑mycin B), the researchers flow‑sorted YFP‑positive cells on the WOLF platform to collect individual cells that had successfully incorporated the constructs of interest. This sorting step enriched for cells expressing the fluorescent reporter, enabling the expansion of defined clonal populations and facilitating downstream sequencing and functional screening across the engineered genetic design space.
