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CRISPR-Cas9 mutagenesis of A549 cells for variants resistant to the ExoU toxin from Pseudomonas aeruginosa

ABSTRACT: CRISPR-Cas9 mutagenesis of A549 cells for variants resistant to the ExoU toxin from Pseudomonas aeruginosa

PROVIDER: PRJNA647377 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12269847.fastq.gz	Fastqsanger.gz
	SRR12269848.fastq.gz	Fastqsanger.gz
	SRR12269849.fastq.gz	Fastqsanger.gz
	SRR12269850.fastq.gz	Fastqsanger.gz

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Saturating mutagenesis with assembled CRISPR-Cas9 ribonucleoprotein complexes.

Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.

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CRISPR-Cas9 mutagenesis of A549 cells for variants resistant to the ExoU toxin from Pseudomonas aeruginosa

Project description:Pseudomonas aeruginosa is a leading cause of nosocomial infection and is responsible for severe acute and chronic infections such as pneumonia, urinary tract infections and lung infections in cystic fibrosis patients. The bacterium possesses a type 3 secretion system whose expression is linked with poor outcomes in both animal models and human patients. In particular, this syringe-like apparatus injects ExoU, the most toxic T3SS effector. However, the mechanism of ExoU in P. aeruginosa’s virulence remains elusive compare to the other T3SS toxins. It was previously suggested that the toxin uses a host trafficking system to reach the plasma membrane and to induce cell necrosis. In this work, we demonstrated that once injected into the cell cytoplasm, ExoU binds to DNAJC5+ vesicles to rapidly reach the plasma membrane.

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