title: Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance.
abstract: The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA-the major gene involved in methicillin resistance-can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.
authors.affiliation: Graduate School of Nanoscience and Technology and ‡Department of Biological Sciences, Korea Advanced Institute of Science and Technology , Daejeon, 305-701, Republic of Korea.
authors.forename: Yoo Kyung
authors.initials: YK
authors.lastname: Kang
authors.affiliation: Graduate School of Nanoscience and Technology and ‡Department of Biological Sciences, Korea Advanced Institute of Science and Technology , Daejeon, 305-701, Republic of Korea.
authors.forename: Kyu
authors.initials: K
authors.lastname: Kwon
authors.affiliation: Graduate School of Nanoscience and Technology and ‡Department of Biological Sciences, Korea Advanced Institute of Science and Technology , Daejeon, 305-701, Republic of Korea.
authors.forename: Jea Sung
authors.initials: JS
authors.lastname: Ryu
authors.affiliation: Graduate School of Nanoscience and Technology and ‡Department of Biological Sciences, Korea Advanced Institute of Science and Technology , Daejeon, 305-701, Republic of Korea.
authors.forename: Ha Neul
authors.initials: HN
authors.lastname: Lee
authors.affiliation: Graduate School of Nanoscience and Technology and ‡Department of Biological Sciences, Korea Advanced Institute of Science and Technology , Daejeon, 305-701, Republic of Korea.
authors.forename: Chankyu
authors.initials: C
authors.lastname: Park
authors.affiliation: Graduate School of Nanoscience and Technology and ‡Department of Biological Sciences, Korea Advanced Institute of Science and Technology , Daejeon, 305-701, Republic of Korea.
authors.forename: Hyun Jung
authors.identifier: 0000-0001-5055-902X
authors.initials: HJ
authors.lastname: Chung
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D016680:Genome, Bacterial; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects / Q000235:genetics*; D011108:Polymers / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000008:administration & dosage* / Q000235:genetics; D013297:Streptococcus pyogenes / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D011108:Polymers; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases