Genome-Wide Gene Regulation by Transcriptional CRISPRa/i Tools in Non-Model Bacteria
Cholpisit Kiattisewee* (firstname.lastname@example.org), Ian D. Faulkner, Jesse G. Zalatan, and James M. Carothers
University of Washington
CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) are modular tools that can regulate gene expression of both heterologous and endogenous genes of microorganisms. The project’s goal is to use CRISPRa/i to build large gene regulatory networks (GRNs) spanning more than 25 genes. This study will establish a new paradigm for genome-wide design and significantly improve the ability to engineer microbes for next-generation bioproduction applications.
CRISPRa, developed from this group, is an emerging tool for transcriptional regulation in bacteria providing the ability to modulate gene expression in trans without direct modification at the DNA target (Dong et al. 2018; Fontana et al. 2020). The team also characterized the rules for effective CRISPRa in Escherichia coli and Pseudomonas putida, potential chassis for aromatic compounds bioproduction, where the rules governing high-functional CRISPRa are portable across organisms (Fontana et al. 2020; Kiattisewee et al. 2021). With incorporation of protospacer adjacent motif–flexible dCas9 proteins, researchers can target almost any endogenous gene in the bacterial genome (Kiattisewee et al. 2022), and by combining with CRISPRi gene repression, gene expression can be fine-tuned by both up- and down-regulation to any desired expression level (Tickman et al. 2021). In this study, researchers have demonstrated that CRISPRa/i tools can be used to control heterologous gene expression for bioproduction of various fine chemicals. The team has also investigated CRISPRa/i of more than 25 endogenous genes related to carbohydrate, amino acids, and fatty acids metabolism. This CRISPRa/i platform should provide the ability to combine heterologous and endogenous gene regulations and further accelerate Design-Build-Test-Learn cycles of strains engineering in non-model bacteria.
Dong, C., et al. 2018. “Synthetic CRISPR-Cas Gene Activators for Transcriptional Reprogramming in Bacteria,” Nature Communications 9(1), 2489. DOI:10.1038/s41467-018-04901-6.
Fontana, J., et al. 2020. “Effective CRISPRa-Mediated Control of Gene Expression in Bacteria Must Overcome Strict Target Site Requirements,” Nature Communications 11(1), 1618. DOI:10.1038/s41467-020-15454-y.
Kiattisewee, C., et al. 2021. “Portable Bacterial CRISPR Transcriptional Activation Enables Metabolic Engineering in Pseudomonas putida,” Metabolic Engineering 66, 283–95. DOI:10.1016/j.ymben.2021.04.002.
Kiattisewee, C., et al. 2022. “Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible dCas9 Variants,” ACS Synthetic Biology 11(12), 4103–12. DOI:10.1021/acssynbio.2c00405.
Tickman, B. I., et al. 2021. “Multi-Layer CRISPRa/i Circuits for Dynamic Genetic Programs in Cell-Free and Bacterial Systems,” Cell Systems 13(3), 215–29.e8. DOI:10.1016/j.cels.2021.10.008.
This research was supported by the U.S. Department of Energy Bioenergy Technologies Office (BETO), grant no. DE-EE0008927 and DOE Office of Science, Biological and Environmental Research (BER) Program, grant no. DE-SC0023091.