Automated Mini-Bioreactors Reveal the Temporal Dynamics and Multi-Omics Responses of CRISPRi Knockdowns in Pseudomonas putida

IF 4.5 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2026-10-16 Epub Date: 2026-09-18 DOI:10.1021/acssynbio.6c00205

Mariana Arango Saavedra, Sara Grassi, Magnus Ganer Jespersen, Catarina Rocha, Viji Kandasamy, Lars Keld Nielsen, Pablo Ivan Nikel, Stefano Donati

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引用次数: 0

Abstract

Characterizing CRISPR interference (CRISPRi) phenotypes presents a fundamental temporal challenge: pre-existing overabundance of target proteins can mask early silencing, requiring extended growth for dilution, yet prolonged repression rapidly selects for escaper mutants. To resolve this, we integrated a tightly regulated CRISPRi system in Pseudomonas putida with an automated mini bioreactor platform operating in turbidostat mode. By maintaining continuous exponential growth, we mapped the exact temporal dynamics of essential gene silencing. We identified a critical observation window between 7.8 and 26.7 h (corresponding to 6.4−23.5 cumulative cell doublings of the uninduced condition) where repression exerts its maximum physiological impact, directly preceding population takeover by target-site mutated escapers. Applying this workflow to the arginine biosynthesis pathway, multi-omics profiling disentangled transient physiological buffering from long-term mutational events, revealing that argH and argG knockdowns trigger highly diverse metabolomic perturbations. This scalable framework overcomes batch culture limitations for CRISPRi experiments, ensuring precise temporal control for accurate phenotypic characterization and reliable functional genomics.

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自动微型生物反应器揭示恶臭假单胞菌CRISPRi敲低的时间动态和多组学响应

表征CRISPR干扰（CRISPRi）表型提出了一个基本的时间挑战：预先存在的过多的靶蛋白可以掩盖早期沉默，需要延长生长以稀释，然而长时间的抑制迅速选择逃逸突变体。为了解决这个问题，我们在恶臭假单胞菌中集成了一个严格调控的CRISPRi系统，并在浊度控制模式下运行了一个自动化的微型生物反应器平台。通过保持持续的指数增长，我们绘制了基本基因沉默的确切时间动态。我们确定了7.8至26.7小时之间的关键观察窗口（对应于未诱导条件下的6.4 - 23.5倍累积细胞），在此抑制发挥其最大的生理影响，直接在目标位点突变逃逸者接管种群之前。将这一工作流程应用于精氨酸生物合成途径，多组学分析从长期突变事件中解开了短暂的生理缓冲，揭示了argH和argG敲低引发高度多样化的代谢组学扰动。这种可扩展的框架克服了CRISPRi实验的批量培养限制，确保了准确的表型表征和可靠的功能基因组学的精确时间控制。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.