Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance
A recent study published in Bioconjugate Chemistry, describes a nonviral genome editing strategy using a polymer-derivatized CRISPR nanocomplex (Cr-Nanocomplex) for targeting antibacterial resistance.
The Cr-Nanocomplex was characterized as small, nano-sized protein-polymer conjugate/RNA complexes, which can be designed to target antibacterial resistance.
The Cr-Nanocomplex was applied to target mecA- the major resistance gene involved in Methicillin-resistant Staphylococcus aureus (MRSA)- and showed that editing of the bacterial genome resulted in greatly reduced growth of MRSA, compared to growth observed for the native complex or conventional lipid-based formulations.
This achievement showed for the first time that covalent modification of the non-viral CRISPR system allows its efficient delivery into cellular targets, and is able to edit the bacterial genome.
Over the past several decades, the overuse of antibiotics has dramatically increased the spread of multidrug-resistant bacteria.
Sustained treatment with antibiotics naturally selects the mutant bacterial clones with resistance acquired by changes in intrinsic expression or horizontal transfer of genes.
The spread of multidrug-resistant bacteria have resulted in more limited treatment options, requiring the use or discovery of more potent drugs.
A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth.
A research team led by Professor Hyun Jung Chung from GSNT introduced a nonviral treatment strategy delivering genome editing material for the targeting of antibacterial resistance.
The research team applied the CRISPR-Cas9 system, recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo.
They utilized polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance.
Nano-sized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage.
Polymer derivatization of the Cas9 protein was expected to enhance their uptake into bacteria, compared to native Cas9 due to the highly cationic property of the polymer, and an increase in polarity of the protein.
Results from the experiment supported the hypothesis that the Cr-Nanocomplex designed to target mecA can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allows for the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations.
Compared to the previously reported noncovalent formulations, the direct conjugation of Cas9 with polymer allowed each single molecule of Cas9 protein to be bound to the carrier material.
This importantly resolves loading efficiency issues found in the previous cases of noncovalent formulations.
Also, since only one or two bPEI molecules of 2,000 Da was conjugated onto each Cas9 molecule, a minimal amount of carrier material can be used, thus helping minimize toxicity/side effects and allow injections at higher dosages.
Since bacteria have a thick cell wall, compared to mammalian cells, maximizing transfection efficiency is critical; most nonviral delivery systems show lower transfection efficiencies than virus-based delivery.
When sufficient delivery and genome editing is achieved, this system can be applied as a target-specific or ‘narrow spectrum’ antimicrobial, potentially useful for treating multidrug-resistant infections in the clinic to prevent drug overuse, while controlling the further spread of antibiotic resistance.
Hyun Jung Chung, her students Yoo Kyung Kang, Jea Sung Ryu, Ha Neul Lee and Chankyu Park, his post-doctoral researcher Kyu Kwon from KAIST appear as authors of this article published in April: Bioconjugate Chem., 28 (4) : 957–967, 2017.
* Lab information : http://nanomedicine.kaist.ac.kr (Lab webpage)