A ‘poly-transfection’ method for rapid, one-pot characterization and optimization of genetic systems
| Autor: | Breanna DiAndreth, Jeremy J Gam, Ross D. Jones, Ron Weiss, Jin Huh |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
animal structures
viruses Gene Expression Computational biology Biology Transfection 03 medical and health sciences 0302 clinical medicine Genetics Animals Humans Gene 030304 developmental biology 0303 health sciences Extramural fungi Genetic systems High-Throughput Screening Assays MicroRNAs embryonic structures Methods Online CRISPR-Cas Systems Classifier (UML) Design space 030217 neurology & neurosurgery |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkz623 |
| Popis: | Biological research is relying on increasingly complex genetic systems and circuits to perform sophisticated operations in living cells. Performing these operations often requires simultaneous delivery of many genes, and optimizing the stoichiometry of these genes can yield drastic improvements in performance. However, sufficiently sampling the large design space of gene expression stoichiometries in mammalian cells using current methods is cumbersome, complex, or expensive. We present a ‘poly-transfection’ method as a simple yet high-throughput alternative that enables comprehensive evaluation of genetic systems in a single, readily-prepared transfection sample. Each cell in a poly-transfection represents an independent measurement at a distinct gene expression stoichiometry, fully leveraging the single-cell nature of transfection experiments. We first benchmark poly-transfection against co-transfection, showing that titration curves for commonly-used regulators agree between the two methods. We then use poly-transfections to efficiently generate new insights, for example in CRISPRa and synthetic miRNA systems. Finally, we use poly-transfection to rapidly engineer a difficult-to-optimize miRNA-based cell classifier for discriminating cancerous cells. One-pot evaluation enabled by poly-transfection accelerates and simplifies the design of genetic systems, providing a new high-information strategy for interrogating biology. |
| Databáze: | OpenAIRE |
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