Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System

<u>C</u>lustered <u>R</u>egularly <u>I</u>nterspaced <u>S</u>hort <u>P</u>alindromic <u>R</u>epeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9

Antibacterial Activity of THAM Trisphenylguanide against Methicillin-Resistant <i>Staphylococcus aureus</i>

<div><p>This study investigated the potential antibacterial activity of three series of compounds synthesized from 12 linear and branched polyamines with 2–8 amino groups, which were substituted to produce the corresponding guanides, biguanides, or phenylguanides, against <i>Acinetobacter baumannii</i>, <i>Enterococcus faecalis</i>, <i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i>. Antibacterial activity was measured for each compound by determining the minimum inhibitory concentration against the bacteria, and the toxicity towards mammalian cells was determined. The most effective compound, THAM trisphenylguanide, was studied in time-to-kill and cytoplasmic leakage assays against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA, USA300) in comparison to chlorhexidine. Preliminary toxicity and MRSA challenge studies in mice were also conducted on this compound. THAM trisphenylguanide showed significant antibacterial activity (MIC ∼1 mg/L) and selectivity against MRSA relative to all the other bacteria examined. In time-to-kill assays it showed increased antimicrobial activity against MRSA versus chlorhexidine. It induced leakage of cytoplasmic content at concentrations that did not reduce cell viability, suggesting the mechanism of action may involve membrane disruption. Using an intraperitoneal mouse model of invasive MRSA disease, THAM trisphenylguanide reduced bacterial burden locally and in deeper tissues. This study has identified a novel guanide compound with selective microbicidal activity against <i>Staphylococcus aureus,</i> including a methicillin-resistant (MRSA) strain.</p></div

Median MICs and MBCs for chlorhexidine and THAM-3ΦG in MHB.

<p>There were no significant differences between MIC and MBC for each <i>S. aureus</i> strain and no significant differences between the two <i>S. aureus</i> strains using 2-tailed unpaired T-tests (p>0.05, n≥7).</p

Time-to-kill assays.

<p>MRSA (a–b) and <i>E. coli</i> (c–d) treated with chlorhexidine (a, c) and THAM-3ФG (b, d). Significant differences between THAM-3ФG and chlorhexidine treatments were only seen at 4×MIC at 4 and 6 h with MRSA (p-value<0.01) and at 1 and 2 h with <i>E. coli</i> (p-value<0.05). P-values were calculated for each time point and between compounds by one-way ANOVA.</p

Structures of chlorhexidine (top) and THAM-3ΦG.

<p>Structures of chlorhexidine (top) and THAM-3ΦG.</p

Cytotoxicities of the guanide, biguanide, phenylguanide derivatives, and the parent amines, against a human breast cancer cell line (MDA-231) and a human keratinocyte cell line (HaCat).

a<p>CC₅₀ is the concentration of compound which killed 50% of the cells in the MTS assay.</p>b<p>Factor by which cytotoxicity was reduced relative to chlorhexidine.</p

THAM-3ΦG reduces MRSA burden in vivo during peritonitis.

<p>Mice were treated with PBS control or with THAM trisphenylguanide immediately after or at one hour post intraperitoneal infection. Bacterial burden was evaluated in the kidney (A) and heart (B); localized infection was assessed with peritoneal lavage fluid (C). The horizontal bar is the mean of the eight mice per treatment group; error bars represent standard error of the mean. Reductions in burden were significant (t-test) in kidneys for both the 1 hr (p = 0.028) and 0 hr (p = 2×10⁻⁶) treatments. Reductions were significant in the heart (p = 4×10⁻⁵) and IP lavage (p = 6×10⁻⁸) only for the 0 hr treatments.</p

Minimum Inhibitory Concentrations of the most active guanide (G) and phenylguanide (ΦG) compounds.

<p>Values are the median of at least 3 determinations. >256 indicates no inhibition at the highest concentration tested. TOAM was insoluble at >64 mg/L.</p