title: Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA.
abstract: CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility. The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg kg-1). Intravenous co-delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing.
authors.affiliation: University of Texas Southwestern Medical Center, Simmons Comprehensive Cancer Center, Department of Biochemistry, Dallas, TX, 75390, USA.
authors.forename: Jason B
authors.identifier: 0000-0002-6587-250X
authors.initials: JB
authors.lastname: Miller
authors.affiliation: University of Texas Southwestern Medical Center, Children's Research Institute, Departments of Pediatrics and Internal Medicine, Dallas, TX, 75390, USA.
authors.forename: Shuyuan
authors.initials: S
authors.lastname: Zhang
authors.affiliation: University of Texas Southwestern Medical Center, Simmons Comprehensive Cancer Center, Department of Biochemistry, Dallas, TX, 75390, USA.
authors.forename: Petra
authors.initials: P
authors.lastname: Kos
authors.affiliation: University of Texas Southwestern Medical Center, Simmons Comprehensive Cancer Center, Department of Biochemistry, Dallas, TX, 75390, USA.
authors.forename: Hu
authors.initials: H
authors.lastname: Xiong
authors.affiliation: University of Texas Southwestern Medical Center, Simmons Comprehensive Cancer Center, Department of Biochemistry, Dallas, TX, 75390, USA.
authors.forename: Kejin
authors.initials: K
authors.lastname: Zhou
authors.affiliation: University of Texas Southwestern Medical Center, Department of Microbiology, Dallas, TX, 75390, USA.
authors.forename: Sofya S
authors.initials: SS
authors.lastname: Perelman
authors.affiliation: University of Texas Southwestern Medical Center, Children's Research Institute, Departments of Pediatrics and Internal Medicine, Dallas, TX, 75390, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhu
authors.affiliation: University of Texas Southwestern Medical Center, Simmons Comprehensive Cancer Center, Department of Biochemistry, Dallas, TX, 75390, USA.
authors.forename: Daniel J
authors.identifier: 0000-0003-3823-1931
authors.initials: DJ
authors.lastname: Siegwart
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D000072669:Gene Editing; D018014:Gene Transfer Techniques; D008055:Lipids / Q000737:chemistry; D015394:Molecular Structure; D053758:Nanoparticles / Q000737:chemistry*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*
chemical_list: D008055:Lipids; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger
keywords: CRISPR/Cas; gene editing; mRNA delivery; nanoparticles; sgRNA delivery