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.

The binary Clostridium (C.) botulinum C2 toxin consists of two non-linked proteins. The proteolytically activated binding/transport subunit C2IIa forms barrel-shaped homoheptamers, which bind to cell surface receptors, mediate endocytosis, and translocate the enzyme subunit C2I into the cytosol of target cells. Here, we investigate whether C2IIa can be harnessed as a transporter for proteins/enzymes fused to polycationic tags, as earlier demonstrated for the related anthrax toxin transport subunit PA₆₃. To test C2IIa-mediated transport in cultured cells, reporter enzymes are generated by fusing different polycationic tags to the N- or C-terminus of other bacterial toxins’ catalytic A subunits. C2IIa as well as PA₆₃ deliver N-terminally polyhistidine-tagged proteins more efficiently compared to C-terminally tagged ones. However, in contrast to PA₆₃, C2IIa does not efficiently deliver polylysine-tagged proteins into the cytosol of target cells. Moreover, untagged enzymes with a native cationic N-terminus are efficiently transported by both C2IIa and PA₆₃. In conclusion, the C2IIa-transporter serves as a transport system for enzymes that harbor positively charged amino acids at their N-terminus. The charge distribution at the N-terminus of cargo proteins and their ability to unfold in the endosome and subsequently refold in the cytosol determine transport feasibility and efficiency.

Chemoselective dual functionalization of proteins has emerged as an invaluable tool to introduce two distinct payloads to proteins, thus greatly expanding their structural and functional repertoire for more advanced biomedical applications. Here, we introduce N-alkylpyridinium reagents as soft electrophiles for chemoselective dual modification of cysteine residues in peptides or proteins via a 1,6-addition reaction. The N-alkylpyridinium derivatives can be synthesized in two reaction steps revealing good water solubility, high labelling efficiency and chemoselectivity towards cysteine over lysine/N-terminal amine residues, even when used in large excess. This reaction can be combined with strain-promoted azide-alkyne click (SPAAC) and inverse-electron-demand Diels−Alder (iEDDA) reactions to achieve dual functionalization of proteins in a sequential simple one-pot reaction. As a proof-of-concept, the Rho-inhibiting enzyme Clostridium botulinum C3 is functionalized with a cancer cell-targeting peptide and a fluorescent dye for the inhibition of specific Rho-mediated intracellular pathways. The high stability, ease of synthesis, fast reaction kinetics, high water-solubility and chemoselectivity make N-alkylpyridinium reagents unique for dual modification of peptides and proteins to increase their functional diversities for medical applications.

Introduction: Supplementation with increased inspired oxygen fractions has been suggested to alleviate the harmful effects of tissue hypoxia during hemorrhagic shock (HS) and traumatic brain injury. However, the utility of therapeutic hyperoxia in critical care is disputed to this day as controversial evidence is available regarding its efficacy. Furthermore, in contrast to its hypoxic counterpart, the effect of hyperoxia on the metabolism of circulating immune cells remains ambiguous. Both stimulating and detrimental effects are possible; the former by providing necessary oxygen supply, the latter by generation of excessive amounts of reactive oxygen species (ROS). To uncover the potential impact of increased oxygen fractions on circulating immune cells during intensive care, we have performed a ¹³C-metabolic flux analysis (MFA) on PBMCs and granulocytes isolated from two long-term, resuscitated models of combined acute subdural hematoma (ASDH) and HS in pigs with and without cardiovascular comorbidity.Methods: Swine underwent resuscitation after 2 h of ASDH and HS up to a maximum of 48 h after HS. Animals received normoxemia (P_aO₂ = 80 – 120 mmHg) or targeted hyperoxemia (P_aO₂ = 200 – 250 mmHg for 24 h after treatment initiation, thereafter P_aO₂ as in the control group). Blood was drawn at time points T1 = after instrumentation, T2 = 24 h post ASDH and HS, and T3 = 48 h post ASDH and HS. PBMCs and granulocytes were isolated from whole blood to perform electron spin resonance spectroscopy, high resolution respirometry and ¹³C-MFA. For the latter, we utilized a parallel tracer approach with 1,2-¹³C₂ glucose, U-¹³C glucose, and U-¹³C glutamine, which covered essential pathways of glucose and glutamine metabolism and supplied redundant data for robust Bayesian estimation. Gas chromatography-mass spectrometry further provided multiple fragments of metabolites which yielded additional labeling information. We obtained precise estimations of the fluxes, their joint credibility intervals, and their relations, and characterized common metabolic patterns with principal component analysis (PCA).Results: ¹³C-MFA indicated a hyperoxia-mediated reduction in tricarboxylic acid (TCA) cycle activity in circulating granulocytes which encompassed fluxes of glutamine uptake, TCA cycle, and oxaloacetate/aspartate supply for biosynthetic processes. We further detected elevated superoxide levels in the swine strain characterized by a hypercholesterolemic phenotype. PCA revealed cell type-specific behavioral patterns of metabolic adaptation in response to ASDH and HS that acted irrespective of swine strains or treatment group.Conclusion: In a model of resuscitated porcine ASDH and HS, we saw that ventilation with increased inspiratory O₂ concentrations (P_aO₂ = 200 – 250 mmHg for 24 h after treatment initiation) did not impact mitochondrial respiration of PBMCs or granulocytes. However, Bayesian ¹³C-MFA results indicated a reduction in TCA cycle activity in granulocytes compared to cells exposed to normoxemia in the same time period. This change in metabolism did not seem to affect granulocytes’ ability to perform phagocytosis or produce superoxide radicals.

Metastatic breast carcinoma (BC) cells are prone to spreading in the bone microenvironment, leading to a vicious cycle between local osteoclast-mediated osteolysis and tumor progression. Therefore, the targeted pharmacological down-modulation of BC cell proliferation as well as osteoclast differentiation and hyperactivity might represent a promising treatment option. We developed a multifunctional peptide nanocarrier combining bioactive EPI-X4 peptides and the Rho-inhibiting C3bot enzyme from Clostridium botulinum. C3bot is preferentially internalized into the cytosol of monocytic cells, including osteoclasts, where it inhibits Rho-mediated signal transduction. However, Rho-mediated cellular processes like migration and cell division can also be inhibited in non-monocytic cells if C3bot is delivered into their cytosol by a nanocarrier. To accomplish this, we designed a supramolecular transporter where one molecule of biotinylated C3bot and three biotinylated entities of the human EPI-X4 peptide-derived CXCR4 antagonist JM173 are assembled on avidin as a central platform. This modular transport system (JM173)₃-Avi-C3 down-modulated osteoclast formation and hyperactivity and delivered the therapeutic cargo C3bot successfully into the cytosol of breast cancer cells, where it inhibited Rho.

We report N-alkylpyridinium reagents for site-selective dual modification of proteins via a regioselective 1,6-thiol addition reaction. The N-alkylpyridinium derivatives can be syn-thesized in two reaction steps and demonstrate high labelling efficiency and chemose-lectivity towards cysteine residues. This reaction is combined with strain-promoted azide-alkyne click and inverse-electron-demand Diels−Alder reactions to achieve dual functionalization of proteins in a sequential one-pot reaction. A Rho-inhibiting enzyme is functionalized with a cancer cell-targeting peptide and a fluorescent dye for successful in vitro uptake imaging and concomitant inhibition of specific Rho-mediated cellular pathways. The ease of synthesis, fast kinetics, high solubility and regioselectivity make N-alkylpyridinium reagents unique for protein/peptide modification to increase their functional diversities for medical applications.