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CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli

ABSTRACT: Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.

ORGANISM(S): Escherichia coli

PROVIDER: GSE143629 | GEO | 2020/01/15

REPOSITORIES: GEO

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CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli

Project description:CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli

| PRJNA601148 | ENA

Mutation of the RNA polymerase M-NM-2-subunit gene promotes hetero-to-homo conversion of M-CM-^_-lactam resistance of methicillin-resistant Staphylococcus aureus

Project description:Three types of phenotypic expression of M-CM-^_-lactam resistance has been reported in MRSA: heterogeneous-, homogeneous-, and Eagle-type resistance. Heterogeneous to homogeneous (hetero-to-homo) conversion of M-CM-^_-lactam resistance is postulated to be caused by a chromosomal mutation (chr*) together with mecA-gene expression. The Eagle-type resistance is a special pattern of chr* expression in the pre-MRSA strain N315 under the strong mecI-gene mediated repression of mecA gene transcription. Here, for the identification of chr*, experiments were conducted using an in-vitro derived homogeneously imipenem-resistant MRSA strain N315M-bM-^HM-^FIPH5 (M-bM-^HM-^FIPH5). The strain was selected with imipenem 8 mg/L from the heterogeneously imipenem-resistant MRSA strain N315M-bM-^HM-^FIP (M-bM-^HM-^FIP). The whole genome sequencing of M-bM-^HM-^FIPH5 revealed the presence of a unique mutation in the rpoB gene, rpoB(N967I), causing the amino-acid (AA) substitution of Asp by Ile at the 967th AA position of the RNA polymerase M-CM-^_ subunit. The effect of the mutation on the phenotypic change was confirmed by constructing and studying the phenotype of the strains H5rpoB(I967N), a M-bM-^HM-^FIPH5-derived strain cured of the rpoB mutation, and N315rpoB(N967I), a N315-derived strain introduced with the mutated rpoB gene. H5rpoB(I967N) regained the hetero-MRSA phenotype, and the mutant strain N315rpoB(N967I) showed an Eagle-type phenotype similar to that of N315h4. Furthermore, subsequent whole genome sequencing revealed that N315h4 also had a missense mutation in the rpoB gene rpoB(R644H). The rpoB mutations caused decreased autolysis, prolonged doubling-time, and tolerance to bactericidal concentrations of methicillin. We concluded that the certain rpoB mutations are chr* responsible for the hetero-to-homo phenotypic conversion of MRSA. We compared the gene expression profiles of the wild-type strain and rpoB mutant (N967I) using a 60mer oligo array.

2013-08-27 | E-GEOD-42218 | biostudies-arrayexpress

Mutation of the RNA polymerase β-subunit gene promotes hetero-to-homo conversion of ß-lactam resistance of methicillin-resistant Staphylococcus aureus

Project description:Three types of phenotypic expression of ß-lactam resistance has been reported in MRSA: heterogeneous-, homogeneous-, and Eagle-type resistance. Heterogeneous to homogeneous (hetero-to-homo) conversion of ß-lactam resistance is postulated to be caused by a chromosomal mutation (chr*) together with mecA-gene expression. The Eagle-type resistance is a special pattern of chr* expression in the pre-MRSA strain N315 under the strong mecI-gene mediated repression of mecA gene transcription. Here, for the identification of chr*, experiments were conducted using an in-vitro derived homogeneously imipenem-resistant MRSA strain N315∆IPH5 (∆IPH5). The strain was selected with imipenem 8 mg/L from the heterogeneously imipenem-resistant MRSA strain N315∆IP (∆IP). The whole genome sequencing of ∆IPH5 revealed the presence of a unique mutation in the rpoB gene, rpoB(N967I), causing the amino-acid (AA) substitution of Asp by Ile at the 967th AA position of the RNA polymerase ß subunit. The effect of the mutation on the phenotypic change was confirmed by constructing and studying the phenotype of the strains H5rpoB(I967N), a ∆IPH5-derived strain cured of the rpoB mutation, and N315rpoB(N967I), a N315-derived strain introduced with the mutated rpoB gene. H5rpoB(I967N) regained the hetero-MRSA phenotype, and the mutant strain N315rpoB(N967I) showed an Eagle-type phenotype similar to that of N315h4. Furthermore, subsequent whole genome sequencing revealed that N315h4 also had a missense mutation in the rpoB gene rpoB(R644H). The rpoB mutations caused decreased autolysis, prolonged doubling-time, and tolerance to bactericidal concentrations of methicillin. We concluded that the certain rpoB mutations are chr* responsible for the hetero-to-homo phenotypic conversion of MRSA.

2013-08-27 | GSE42218 | GEO

Identifying the fitness cost of rifampicin-resistance in Mycobacterium tuberculosis through RNAseq profiling.

Project description:The experiment was designed to infer the fitness cost of rifampicin-resistance in Mycobacterium tuberculosis through expression analysis. The approach relied on: 1. Tracking the expression changes occurring as a result of the rifampicin-resistance conferring mutation Ser450Leu in RpoB and subsequent gain of compensatory mutation Leu516Pro in RpoC. The hypothesis was that any cost-incurring expressional changes would be reversed in the presence of compensatory mutations. The strains in this set were described before here: PMID 22179134. 2. Comparing the impact of the same rifampicin-resistance mutation (RpoB Ser450Leu) in five different genetic backgrounds. Here the comparison was solely between RpoB Ser450Leu and their cognate drug susceptible wild type ancestor. One of the strain pairs is the same as in point 1. above.

2018-11-18 | E-MTAB-7359 | biostudies-arrayexpress

High-efficiency RNA cloning enables quantitative measurement of miRNA expression by deep sequencing

Project description:MiRNA-mediated regulation depends on the stoichiometry between miRNAs and their mRNA targets. To decipher dynamic function of this complex layer, it is critical to characterize individual miRNA species within a specific cellular context. Small RNA cloning followed by deep sequencing is uniquely positioned as a genome-wide profiling method to quantify miRNA expression with potentially unlimited dynamic range and provide single-nucleotide resolution for precise miRNA classification and de novo discovery. However, significant biases introduced by RNA ligation steps in the current RNA cloning protocol often lead to inaccurate miRNA quantification by >1000-fold deviation. As a result, it has greatly hindered the broad application of this method. Here we report a highly efficient RNA cloning method that achieves over 90% efficiency for both 5’ and 3’ ligations with diverse small RNA substrates. When applied to a pool of either equimolar or differentially mixed synthetic miRNAs, the deviation of the cloning frequency for each miRNA is minimized to less than 2-fold of the anticipated value. By using samples obtained from multiple tissues and cells, we further demonstrate the accurate quantification of miRNA expression over a dynamic range of four orders of magnitude. Our results also reveal that most cistronic miRNAs are expressed at similar levels and, in each cell population, miRNAs repress their cognate targets in a dosage dependent manner. Collectively, our high-efficiency RNA cloning method combining with deep sequencing establishes a cost-effective approach for accurate genome-wide miRNA profiling. We designed an artificial system composed of synthetic miRNAs for benchmarking biases in small RNA cDNA cloning for NGS.

2013-09-18 | E-GEOD-47858 | biostudies-arrayexpress

Measuring gene expression in serially-evolved E. coli BW25113 strains in M9+glucose and M9+glucose+rifampicin

Project description:We measured steady-state E. coli transcript levels in 1) a pykF(C8Y) mutant, 2) a rpoB(T1037P) mutant, and 3) five rifampicin-resistant rpoB mutant strains selected from each of these single mutants, in the presence and absence of rifampicin.

2020-10-23 | GSE136977 | GEO

A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins

Project description:Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and – for AcrIIA5 – indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization.

2024-03-01 | GSE254101 | GEO

A mutation of RNA polymerase β’ subunit (RpoC) converts heterogeneously vancomycin-intermediate S. aureus (hVISA) into ‘slow-VISA’ (sVISA)

Project description:Various mutations in the rpoB gene, which encodes the RNA polymerase beta subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediate S. aureus (VISA) and hetero-VISA (hVISA) strains. We reported that rpoB mutations are also linked to the expression of the recently found slow VISA (sVISA) phenotype (Saito et al 2014 AAC). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of rpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (72.2 min) and high vncomycin MIC (16 mg/L). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of slow growth rate, cell wall thickness, and reduced autolysis. Whole genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation F562L in rpoC. Introduction of the wild-type rpoC gene using multi-copy plasmid resolved the sVISA phenotype of V6-5, indicating that rpoC(P440L) mutation expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated additional 10 revertants to hVISA and VISA. In subsequent whole genome analysis, we identified compensatory mutations in the genes of three distinct functional categories; rpoC gene itself as regulatory mutations, peptidoglycan biosynthesis genes, and relQ which is involved in stringent response. It appears that rpoC(P440L) mutation causes sVISA phenotype by augmenting cell-wall peptidoglycan synthesis, and through the control of stringent response. 9 analysis using 60 mer-oligo microarray, 4 rpoC mutant, 2 relQ mutant, 2 mutations associated with the peptidoglycan, 1 wild-type strain

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Biological cost of rifampicin resistance in Helicobacter pylori

Project description:The present study was aimed at analyzing (i) the biological cost of RNA polymerase (rpoB) mutations conferring rifampin resistance on H.pylori, (ii) the relationship between the cost of rpoB mutations and the chromosomal mutaion, (iii) the relationship between the cost of rpoB mutations and the transcription profile of sensitive and resistantrif strains of H.pylori (iv) and rpoB mutations in view of the possible fitness burden associated with resistance to another antibiotics.

2010-11-30 | GSE25333 | GEO

Mutational scanning pinpoints distinct binding sites of key ATGL regulators in lipolysis

Project description:ATGL is the key enzyme in intracellular lipolysis playing a critical role in metabolic and cardiovascular diseases. ATGL is tightly regulated through a known set of protein-protein interaction partners with activating or inhibiting functions in control of lipolysis. However, the binding mode and protein interaction sites of ATGL and its partners are unknown. Using deep mutational protein interaction perturbation scanning we generated comprehensive profiles of single amino acid variants effecting the interactions of ATGL with its regulatory partners: CGI-58, G0S2, PLIN1, PLIN5 and CIDEC. Twenty-three ATGL variants gave a specific interaction perturbation pattern when validated in co-immunoprecipitation experiments in mammalian cells. We identified and characterized eleven, highly selective ATGL “switch” mutations which affect the interaction of one of the five partners without affecting the others. Switch mutations thus provided distinct interaction determinants for ATGL’s key regulatory proteins at an amino acid resolution. When tested for triglyceride hydrolase activity in vitro and lipolysis in cells, the activity patterns of the ATGL switch variants traced to their protein interaction profile. In the context of structural data, the integration of variant binding and activity profiles provided important insights into lipolysis regulation and the impact of mutations in human disease.

2024-03-14 | PXD049436 | Pride

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