Predictive genome-wide CRISPRi-guided gene downregulation for enhanced bioproduction

ABSTRACT: CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, conventional approaches for identifying target genes for CRISPRi-mediated downregulation have largely relied on heuristic methods and trial and error, which are labor-intensive and time-consuming. Additionally, the ability to achieve multigene knockdowns is limited by the constraints of molecular cloning techniques required for building multiplexed CRISPRi systems. In this study, we describe two novel methodologies FluxRETAP (Flux-REaction TArget Prioritization, a Genome-Scale Modeling Technique) and VAMMPIRE (a Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation). Together, these methods establish a systematic and versatile approach for metabolic pathway optimization by elucidating target genes for knockdown using CRISPRi, then facilitating the design of multiplexed sgRNAs to improve the production of a sustainable aviation fuel precursor isoprenol. Our investigation demonstrates the efficacy of FluxRETAP in predicting knockdown target genes that enable improved biosynthesis of isoprenol. Furthermore, VAMMPIRE streamlines the design and construction of CRISPRi arrays of up to five sgRNAs in a single step with ease and high accuracy. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.

INSTRUMENT(S):

ORGANISM(S): Pseudomonas Putida Kt2440

SUBMITTER: Christopher Petzold  

LAB HEAD: Christopher J. Petzold

PROVIDER: PXD062697 | Pride | 2026-10-15

REPOSITORIES: Pride