CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility.[
1] The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility.[
1e] 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.