CRISPR/Cas9-based genome editing for simultaneous interference with gene expression and protein stability.

Interference with genes is the foundation of reverse genetics and is key to manipulation of living cells for biomedical and biotechnological applications. However, classical genetic knockout and transcriptional knockdown technologies have different drawbacks and offer no control over existing protein levels. Here, we describe an efficient genome editing approach that affects specific protein abundances by changing the rates of both RNA synthesis and protein degradation, based on the two cross-kingdom control mechanisms CRISPRi and the N-end rule for protein stability. In addition, our approach demonstrates that CRISPRi efficiency is dependent on endogenous gene expression levels. The method has broad applications in e.g. study of essential genes and antibiotics discovery.

A versatile one-step CRISPR-Cas9 based approach to plasmid-curing.

BACKGROUND: Plasmids are widely used and essential tools in molecular biology. However, plasmids often impose a metabolic burden and are only temporarily useful for genetic engineering, bio-sensing and characterization purposes. While numerous techniques for genetic manipulation exist, a universal tool enabling rapid removal of plasmids from bacterial cells is lacking. RESULTS: Based on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have constructed a universal plasmid-curing system (pFREE) and developed a one-step protocol and PCR procedure that allow for identification of plasmid-free clones within 24 h. While the context of the targeted replicons affects efficiency, we obtained curing efficiencies between 40 and 100% for the plasmids most widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both Escherichia coli and the promising cell factory chassis Pseudomonas putida. CONCLUSION: As a fast and freely available plasmid-curing system, targeting virtually all vectors used for cloning and expression purposes, we believe that pFREE has the potential to eliminate the need for individualized vector suicide solutions in molecular biology. We envision the application of pFREE to be especially useful in methodologies involving multiple plasmids, used sequentially or simultaneously, which are becoming increasingly popular for genome editing or combinatorial pathway engineering.

Restoring Global Gene Regulation through Experimental Evolution Uncovers a NAP (Nucleoid-Associated Protein)-Like Behavior of Crp/Cap.

How do hierarchical gene regulation networks evolve in bacteria? Nucleoid-associated proteins (NAPs) influence the overall structure of bacterial genomes, sigma factors and global transcription factors (TFs) control thousands of genes, and many operons are regulated by highly specific TFs that in turn are controlled allosterically by cellular metabolites. These regulatory hierarchies have been shaped by millions of years of evolution to optimize fitness in response to changing environmental conditions, but it is unclear how NAPs and TFs relate and have evolved together. Cyclic AMP (cAMP) receptor protein (Crp) is the paradigmatic global TF in Escherichia coli, and here we report that mutations in the topA gene compensate for loss of cAMP, showing that the interplay between Crp and the supercoiling status of promoters is key to global stress response. Furthermore, we observed an effect of apoCrp on gene expression in the absence of its effector cAMP. This provides support for the proposed NAP-like role for Crp, suggesting that it represents an intermediate point in the evolution of a ligand-controlled TF from a NAP. IMPORTANCE Here we report that mutations in the topA gene compensate for loss of cAMP, showing that the interplay between Crp and the supercoiling status of promoters is key to global stress response. Furthermore, we observed an effect of apoCrp on gene expression in the absence of its effector cAMP. This provides support for the proposed NAP-like role for Crp, suggesting that it represents an intermediate point in the evolution of a ligand-controlled TF from a NAP.

Temporal evolution of master regulator Crp identifies pyrimidines as catabolite modulator factors.

The evolution of microorganisms often involves changes of unclear relevance, such as transient phenotypes and sequential development of multiple adaptive mutations in hotspot genes. Previously, we showed that ageing colonies of an E. coli mutant unable to produce cAMP when grown on maltose, accumulated mutations in the crp gene (encoding a global transcription factor) and in genes involved in pyrimidine metabolism such as cmk; combined mutations in both crp and cmk enabled fermentation of maltose (which usually requires cAMP-mediated Crp activation for catabolic pathway expression). Here, we study the sequential generation of hotspot mutations in those genes, and uncover a regulatory role of pyrimidine nucleosides in carbon catabolism. Cytidine binds to the cytidine regulator CytR, modifies the expression of sigma factor 32 (RpoH), and thereby impacts global gene expression. In addition, cytidine binds and activates a Crp mutant directly, thus modulating catabolic pathway expression, and could be the catabolite modulating factor whose existence was suggested by Jacques Monod and colleagues in 1976. Therefore, transcription factor Crp appears to work in concert with CytR and RpoH, serving a dual role in sensing both carbon availability and metabolic flux towards DNA and RNA. Our findings show how certain alterations in metabolite concentrations (associated with colony ageing and/or due to mutations in metabolic or regulatory genes) can drive the evolution in non-growing cells.

Mutations in the Global Transcription Factor CRP/CAP: Insights from Experimental Evolution and Deep Sequencing.

The Escherichia coli cyclic AMP receptor protein (CRP or catabolite activator protein, CAP) provides a textbook example of bacterial transcriptional regulation and is one of the best studied transcription factors in biology. For almost five decades a large number of mutants, evolved in vivo or engineered in vitro, have shed light on the molecular structure and mechanism of CRP. Here, we review previous work, providing an overview of studies describing the isolation of CRP mutants. Furthermore, we present new data on deep sequencing of different bacterial populations that have evolved under selective pressure that strongly favors mutations in the crp locus. Our new approach identifies more than 100 new CRP mutations and paves the way for a deeper understanding of this fascinating bacterial master regulator.

Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability.

In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest. The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool.

Standardized Cloning and Curing of Plasmids.

Plasmids are highly useful tools for studying living cells and for heterologous expression of genes and pathways in cell factories. Standardized tools and operating procedures for handling such DNA vectors are core principles in synthetic biology. Here, we describe protocols for molecular cloning and exchange of genetic parts in the Standard European Vectors Architecture (SEVA) vector system. Additionally, to facilitate rapid testing and iterative bioengineering using different vector designs, we provide a one-step protocol for a universal CRISPR-Cas9-based plasmid curing system (pFREE) and demonstrate the application of this system to cure SEVA constructs (all vectors are available at SEVA/Addgene).

Increasing the permeability of Escherichia coli using MAC13243.

The outer membrane of gram-negative bacteria is a permeability barrier that prevents the efficient uptake of molecules with large scaffolds. As a consequence, a number of antibiotic classes are ineffective against gram-negative strains. Herein we carried out a high throughput screen for small molecules that make the outer membrane of Escherichia coli more permeable. We identified MAC13243, an inhibitor of the periplasmic chaperone LolA that traffics lipoproteins from the inner to the outer membrane. We observed that cells were (1) more permeable to the fluorescent probe 1-N-phenylnapthylamine, and (2) more susceptible to large-scaffold antibiotics when sub-inhibitory concentrations of MAC13243 were used. To exclude the possibility that the permeability was caused by an off-target effect, we genetically reconstructed the MAC13243-phenotype by depleting LolA levels using the CRISPRi system.