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

Jason B. Miller, Shuyuan Zhang, Petra Kos, Hu Xiong, Kejin Zhou, Sofya S. Perelman, Hao Zhu, Daniel J. Siegwart

Research output: Contribution to journalArticle

163 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1059-1063
Number of pages5
JournalAngewandte Chemie - International Edition
Volume56
Issue number4
DOIs
StatePublished - Jan 19 2017

Keywords

  • CRISPR/Cas
  • gene editing
  • mRNA delivery
  • nanoparticles
  • sgRNA delivery

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)

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