Comprehensive Genome-Wide CRISPR Interference Library for High-Throughput Functional Genomic Studies in Pseudomonas putida
Jacob Fenster1,2, Andrew Hren1,2, Margaret Spangler2,3, Bill Alexander2,4, Jerome Fox1, Jeffrey C. Cameron1,2, Carrie A. Eckert2,4* (firstname.lastname@example.org), and Gerald A. Tuskan2,4
1University of Colorado–Boulder, CO; 2Center for Bioenergy Innovation, Oak Ridge National Laboratory; 3University of Tennessee–Knoxville; and 4Oak Ridge National Laboratory
The Center for Bioenergy Innovation (CBI) vision is to accelerate domestication of bioenergy-relevant, nonmodel plants and microbes to enable high-impact innovations along the bioenergy and bioproduct supply chain while focusing on sustainable aviation fuels (SAF). CBI has four overarching innovation targets: (1) Develop sustainable, process-advantaged biomass feedstocks, (2) Refine consolidated bioprocessing with cotreatment to create fermentation intermediates, (3) Advance lignin valorization for biobased products and aviation fuel feedstocks, and (4) Improve catalytic upgrading for SAF blendstocks certification.
The current paradigm for microbial engineering utilizes the design-build-test-learn cycle (DBTL), where production chassis are iteratively engineered based on previous findings toward the development of more robust production strains. Advanced genetic tools can accelerate this cycle, enabling the construction of strains with desirable phenotypes at a faster pace. With the advent of next-generation sequencing and CRISPR-Cas9-mediated genome editing, high-throughput genome-wide functional genomics experiments are now possible, enabling the study of hundreds of thousands of mutations in a single experiment. This work adapts existing knowledge gained using these high-throughput CRISPR-Cas9 technologies in model organisms to the promising nonmodel lignin-degrading soil bacterium Pseudomonas putida KT2440 to enable novel experimental approaches in this host for genotype-phenotype discovery for engineering efforts.
The team has optimized CRISPR-interference (CRISPRi) for P. putida by screening inducible promoter systems that express catalytically dead spCas9, a variant of Cas9 that can still associate with guide RNA (gRNA) and bind targeted sites in the promoter or 5’ end of a gene, resulting in knockdown of gene expression. Researchers have quantified the dynamic range of repression by targeting a genomically integrated fluorescent reporter as well as key metabolic genes that compete with pathways to target products to increase titers (Fenster et al. 2022). In collaboration with the DOE Joint Genome Institute (JGI), team members have generated genome-wide gRNA libraries to identify functional guides for gene editing and repression via CRISPRi to expand this system for genome-scale studies. For CRISPRi, researchers designed a 78,932-member library targeting each gene in the genome with 10-15 gRNAs per gene as well as 798 non-targeting gRNAs (1%) as internal controls. This library was transformed into P. putida and grown under varied growth conditions including glucose, acetate, and 10- and 50-mM p-coumaric acid, one of the major components of lignin hydrolysates that can be valorized by P. putida. Following selective growth, Illumina sequencing was performed where gRNA plasmids serve as barcodes and are used to compare pre- and post-selection to determine enrichment and drop out in the population to non-targeting gRNAs in the populations as controls. These data represent a wealth of new knowledge, currently uncovering essential genes (including a number that are of unknown function) as well as gene knockdowns that enrich or inhibit growth under each of the selective conditions. Validation of these identified targets will lead to discovery of new gene functions and optimization of gene expression in production strains to further expand engineering efforts and continue to accelerate the DBTL cycle in support of CBI research needs.
Fenster, J. A., et al. 2022. “Dynamic and Single Cell Characterization of a CRISPR-Interference Toolset in Pseudomonas putida KT2440 for β-ketoadipate Production from p-coumarate,” Metabolic Engineering Communications 15, e00204. DOI:10.1016/j.mec.2022.e00204.
Funding was provided by the Center for Bioenergy Innovation (CBI) led by Oak Ridge National Laboratory. CBI is funded as a U.S. Department of Energy Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science under FWP ERKP886. Oak Ridge National Laboratory is managed by UT- Battelle, LLC for the U.S. Department of Energy under contract no. DE-AC05-00OR22725.