Non‐Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co‐Delivery of Cas9 mRNA and sgRNA

TLDR

CRISPR/Cas is a revolutionary gene editing technology with wide‑ranging utility. The study aims to develop a safe, non‑viral delivery system for CRISPR/Cas components to enhance therapeutic potential. The authors synthesized zwitterionic amino lipids (ZALs) that can co‑deliver long RNAs such as Cas9 mRNA and sgRNAs. ZAL nanoparticles achieved >90 % protein knockdown with low sgRNA doses, sustained 95 % suppression via permanent DNA editing, high protein expression from low mRNA doses in vitro and in vivo, and successful co‑delivery of Cas9 mRNA and sgLoxP to induce floxed tdTomato expression in mouse liver, kidneys, and lungs, demonstrating their potential for safe, non‑viral gene editing.

Abstract

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 n m ) 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.