Automation and Expansion of EMMA Assembly for Fast-Tracking Mammalian System Engineering.

ABSTRACT

With applications from functional genomics to the production of therapeutic biologics, libraries of mammalian expression vectors have become a cornerstone of modern biological investigation and engineering. Multiple modular vector platforms facilitate the rapid design and assembly of vectors. However, such systems approach a technical bottleneck when a library of bespoke vectors is required. Utilizing the flexibility and robustness of the Extensible Mammalian Modular Assembly (EMMA) toolkit, we present an automated workflow for the library-scale design, assembly, and verification of mammalian expression vectors. Vector design is simplified using our EMMA computer-aided design tool (EMMA-CAD), while the precision and speed of acoustic droplet ejection technology are applied in vector assembly. Our pipeline facilitates significant reductions in both reagent usage and researcher hands-on time compared with manual assembly, as shown by system Q-metrics. To demonstrate automated EMMA performance, we compiled a library of 48 distinct plasmid vectors encoding either CRISPR interference or activation modalities. Characterization of the workflow parameters shows that high assembly efficiency is maintained across vectors of various sizes and design complexities. Our system also performs strongly compared with manual assembly efficiency benchmarks. Alongside our automated pipeline, we present a straightforward strategy for integrating gRNA and Cas modules into the EMMA platform, enabling the design and manufacture of valuable genome editing resources.