(152e) Crispr-Cas9 Based Transcription Inactivation and Elevation System for Multiplex and Tunable Control of Metabolic Flux in One Step

Feng, X., Virginia Polytechnic Institute and State University
Sheng, J., Virginia Polytechnic Institute and State University
Guo, W., Virginia Polytechnic Institute and State University
Ash, C., Virginia Polytechnic Institute and State University
The success of the metabolic engineering requires efficient regulation of native and heterologous genetic elements in multi-enzyme pathways. Therefore, it is crucial to achieve the multiplex and tunable control of pathway activities. Previous approaches to achieve such goal often rely on engineering DNA-binding proteins (e.g., transcription factors), which is a multistep biomolecular processes for construction and optimization of function, and could only be applied to a small amount of genes. Recently, CRISPR-Cas9 system of Streptococcus pyogenes was proved to be capable of either activating or repressing multiple genes in prokaryotic and eukaryotic systems. In this study, we designed a CRISpr-cas9 based Transcription Inactivation aNd Elevation System (CRISTINES) to achieve multiplex and tunable control of metabolic flux in one step. In general, we found that by selectively designing and positioning guide RNAs with base-pairing complementarity to target DNA sites, a synthetic dCas9 system fused with VP64 (a commonly used eukaryotic transcription activator domain) could activate and repress the expressions of multiple genes at the same time. In order to uncover the designing principle for the guide RNAs to achieve the precise transcriptional regulation, we next systemically screened a library of guide RNAs that targeted on over 400 different sites on multiple fluorescent protein-encoding genes, followed by measuring the gene expression (i.e., fluorescent signal) and developing a regression model to accurately simulate the quantitative relation between the gene expression and the guide RNA design. Finally, the functionalities of CRISTINES were confirmed in the violacein pathway by simultaneously tuning the expression level of multiple genes to control synthetic flux towards the production of various chemicals.