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

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.

从功能基因组学到治疗性生物制品生产，哺乳动物表达载体文库已成为现代生物学研究与工程的核心基石。多种模块化载体平台可实现载体的快速设计与组装，但当需要构建定制化载体文库时，这类系统往往会遭遇技术瓶颈。借助可扩展哺乳动物模块化组装（Extensible Mammalian Modular Assembly，简称EMMA）工具包的灵活性与稳定性，我们开发了一套可实现哺乳动物表达载体文库规模的设计、组装与验证的自动化工作流程。载体设计可通过自研的EMMA计算机辅助设计工具（EMMA-CAD）实现简化，而载体组装环节则应用了声滴喷射技术的高精度与高速特性。正如系统Q指标（Q-metrics）所验证的，与手动组装相比，本工作流程可大幅减少试剂消耗量与研究人员的实操耗时。为验证自动化EMMA系统的性能，我们构建了包含48种不同质粒载体的文库，这些载体分别编码CRISPR干扰（CRISPR interference）或CRISPR激活（CRISPR activation）功能模块。对工作流程参数的表征分析表明，无论载体尺寸与设计复杂度如何，本系统均可保持较高的组装效率。相较于手动组装的效率基准值，本系统同样表现优异。除本自动化工作流程外，我们还提出了一种将向导RNA（guide RNA, gRNA）与Cas蛋白模块整合至EMMA平台的简便策略，可用于设计与制备极具应用价值的基因组编辑资源。