Dataset Information

CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin.

ABSTRACT: Translating the CRISPR/Cas9 genome editing technology into clinics is still hampered by rather unspecific, unsafe and/or inconvenient approaches for the delivery of its main components - the Cas9 endonuclease and a guide RNA - into cells. Here, we describe the development of a novel transient and non-viral Cas9 delivery strategy based on the translocation machinery of the Bacillus anthracis anthrax toxin, PA (protective antigen). We show that Cas9 variants fused to the N-terminus of the lethal factor or to a hexahistidine tag are shuttled through channels formed by PA into the cytosol of human cells. As proof-of-principle, we applied our new approach, denoted as CRISPA, to knock out lipolysis-stimulated lipoprotein receptor (LSR) in the human colon cancer cell line HCT116 and green-fluorescent protein (GFP) in human embryonic kidney 293T cells stably expressing GFP. Notably, we confirmed that the transporter PA can be adapted to recognize specific host cell-surface receptor proteins and may be optimized for cell type-selective delivery of Cas9. Altogether, CRISPA provides a novel, transient and non-viral way to deliver Cas9 into specific cells. Thus, this system is an additional step towards safe translation of the CRISPR/Cas9 technology into clinics.

SUBMITTER: Hirschenberger M

PROVIDER: S-EPMC8558532 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin.

Hirschenberger Maximilian M Stadler Nicole N Fellermann Maximilian M Sparrer Konstantin M J KMJ Kirchhoff Frank F Barth Holger H Papatheodorou Panagiotis P

Frontiers in pharmacology 20211018

Translating the CRISPR/Cas9 genome editing technology into clinics is still hampered by rather unspecific, unsafe and/or inconvenient approaches for the delivery of its main components - the Cas9 endonuclease and a guide RNA - into cells. Here, we describe the development of a novel transient and non-viral Cas9 delivery strategy based on the translocation machinery of the <i>Bacillus anthracis</i> anthrax toxin, PA (protective antigen). We show that Cas9 variants fused to the N-terminus of the l ...[more]

PMID: 34733166

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Project description:Bacillus anthracis causes anthrax through a combination of bacterial infection and toxemia. As a major virulence factor of B. anthracis, anthrax lethal toxin (LT) is a zinc-dependent metalloproteinase, exerting its cytotoxicity through proteolytic cleavage of the mitogen-activated protein kinase kinases, thereby shutting down the MAPK pathways. Anthrax lethal toxin induces host lethality mostly by targeting the cardiovascular system. Although the enzymatic activity and the molecular targets of LT have long been known, the detailed mechanisms underlying cellular/tissue/organ toxicity are still poorly understood. In this work, we sought to investigate the mechanism of LT-induced cellular damage in the cardiovascular system. We demonstrate for the first time that anthrax lethal toxin has potent inhibitory effects on the central metabolism of cardiomyocytes and endothelial cells. This is likely due to the observed downregulating of c-Myc expression through the toxin-induced inhibition of the ERK pathway. Since c-Myc is a master transcription factor controlling the expression of many rate-limiting metabolic enzymes in glycolysis and the tricarboxylic acid cycle, LT's downregulation of c-Myc may lead to the observed bioenergetic collapse, particularly, in cardiomyocytes. Since cardiac cell contraction requires continuous production of large amounts of ATP, potent inhibition of the bioenergetics of cardiomyocytes would be incompatible with life. Thus, LT-induced lethality through targeting cardiomyocytes and endothelial cells appears to be a consequence of a bioenergetic collapse, likely due to the toxin's potent inhibitory activity on the MEK-ERK-c-Myc-metabolic/bioenergetic axis within these target cells of cardiovascular system.IMPORTANCEAnthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis, the causative pathogen of anthrax disease. Anthrax lethal toxin is a metalloproteinase that cleaves and inactivates MEKs, thereby shutting down MAPK pathways, leading to host mortality primarily through targeting of the cardiovascular system. However, the detailed mechanisms underlying the toxin's cellular and tissue toxicity are still poorly understood. Here, we found that anthrax lethal toxin has potent inhibitory activity on glycolysis and oxidative phosphorylation of cardiomyocytes and endothelial cells. These effects appear to be the consequence of downregulation of c-Myc, a master transcription factor that controls many rate-limiting enzymes of glycolysis and the tricarboxylic acid cycle. With the high demand on energy for cardiac contraction, the potent inhibition of cardiomyocyte metabolism by LT would be incompatible with life. This work provides critical insights into why the cardiovascular system is the major in vivo target of LT-induced lethality.

Dataset Information

CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin.

Publications

CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin.

Similar Datasets

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