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.

Keywords: CRISPR/Cas; gene editing; mRNA delivery; nanoparticles; sgRNA delivery.

Fig. 1. ZNPs enable permanent CRISPR/Cas-mediated DNA editing

(A) Sequence specific silencing of luciferase by siRNA (9 nM) and editing by sgRNA (7 nM) in HeLa-Luc-Cas9 cells. N = 4 ± stdev, **** p < 0.0001 (B) Kinetically, silencing with siRNA is transient while sgRNA delivery results in permanent loss of luciferase signal after 2 days. (C) Sequence specific editing of luciferase was confirmed by the Surveyor assay. (D) The chemical structure of ZA3-Ep10.

Fig. 2

ZALs were designed to increase molecular interactions with longer RNAs by combining the chemical and structural roles of zwitterionic lipids and cationic lipids into a single lipid compound. High efficiency reactions provided access to a library of unique charge unbalanced lipids.

Fig. 3. ZNPs enable delivery of long RNAs both in vitro and in vivo

(A) ZA3-Ep10 ZNPs (ZAL:cholesterol:PEG-lipid = 100:77:1 (mol); ZAL:RNA = 7.5:1 (wt)) are uniform for both sgRNA and mRNA. (B) ZA3-Ep10 sgRNA ZNPs show dose-responsive Luc editing in HeLa-Luc-Cas9 cells. ZA3-Ep10 ZNPs can also deliver (C) mCherry mRNA (18h) and (D) luciferase mRNA (24h) to IGROV1 cells. (E) In vivo luciferase expression was achieved by systemic i.v. administration of ZA3-Ep10 Luc mRNA ZNPs (24h). Bioluminescence imaging both in vivo (E, athymic nude mice, 1 mg/kg) and ex vivo (F, C57BL/6 mice, 4 mg/kg) revealed expression of luciferase in liver, lung and spleen tissue.

Fig. 4. ZNPs enable co-delivery of Cas9 mRNA and sgRNA for CRISPR/Cas editing

(A) The kinetics of mRNA and protein expression after ZNP delivery of Cas9 mRNA (0.48 ng/mL mRNA) to A549-Luc cells. Cas9 mRNA levels (A red curve) and protein expression (A black curve, B) were measured over time. (C) ZNPs enable dose responsive expression of Cas9, detectable as low as 0.05 μg/mL delivered mRNA. (D) Surveyor confirmed editing of the luciferase target at mRNA:sgRNA ratios of 3:1 or higher (wt). Co-delivery of Cas9 mRNA and sgCtrl showed no editing (Fig. S21).

Fig. 5. ZNPs enabled non-viral CRISPR/Cas editing in vivo

(A) Schematic representation shows that co-delivery of Cas9 mRNA and sgLoxP deletes the stop cassette and activates downstream tdTomato protein. (B) After administration of ZNPs encapsulating Cas9 mRNA:sgRNA (4:1, wt) at 5 mg/kg total RNA, tdTomato fluorescence was detected in the liver and kidney upon whole organ ex vivo imaging. (C) Confocal fluorescence microscopy of tissue sections showed tdTomato positive cells in liver, lung, and kidneys. Scale bars = 50 μm).