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 sequence elements arranged across multi-kb length scales. Overcoming this barrier could accelerate synthetic biology; by screening diverse gene circuit designs, "composition-to-function" mappings could be created that reveal genetic part composability rules and enable rapid identification of behavior-optimized variants. Here, we introduce CLASSIC, a generalizable genetic screening platform that combines long- and short-read next-generation sequencing (NGS) modalities to quantitatively assess pooled libraries of DNA constructs of arbitrary length. We show that CLASSIC can measure expression profiles of >105drug-inducible gene circuit designs (ranging from 6-9 kb) in a single experiment in human cells. Using statistical inference and machine learning (ML) approaches, we demonstrate that data obtained with CLASSIC enables predictive modeling of an entire circuit design landscape, offering critical insight into underlying design principles. Our work shows that by expanding the throughput and understanding gained with each design-build-test-learn (DBTL) cycle, CLASSIC dramatically augments the pace and scale of synthetic biology and establishes an experimental basis for data-driven design of complex genetic systems.

With the widespread use of genomic editing tools like CRISPR/Cas9, scientists have gained the ability to modify a host genome with high precision. Although these technological advancements have improved throughout the years, there is still a need for better methods to build more sophisticated multi‐part biological circuits. For this reason, we have employed the use of a genomic integration technique, landing pads, which utilized site‐specific recombinases to insert relatively large DNA assemblies into a well‐defined location the host genome. Using the CRISPR/Cas9 system, we knocked‐in an 8kb landing pad repair template into the AAVSl safe harbor locus of HEK293T cells. After single‐cell sorting and verifying the genotype of our clonal cell line, we used the BxB1 recombinase to integrate a destination plasmid into the landing pad site. Using microscopy, flow cytometry, and PCR genotyping, we demonstrate almost complete replacement of the original landing pad template with our destination vector species within 12 days. Here we present an approach that enables the rapid creation of mammalian cell lines harboring complex, multi‐part regulatory circuits at single copy. This platform allows for precise and tunable control of gene expression profiles, providing an excellent opportunity to engineer safer and more sophisticated cell‐‐based therapies. Support or Funding Information National Institute of General Medical Sciences of the National Institutes of Health under linked Award Numbers SC1GM111172, RL5GM118969, TL4GM118971, UL1GM118970, and 5G12MD007592.

Synthetic refactoring makes naturally occurring regulatory systems more amenable to manipulation by removing or recoding their natural genetic complexity. Shaw et al. apply this technique to the yeast mating response pathway, creating a simplified, highly engineerable signaling module that can be used to construct precisely optimized, application-specific GPCR biosensors.