Nonviral Gold Nanoparticle‐Mediated Delivery of CRISPR‐Cas9 Ribonucleoprotein and Long DNA Transgenes Into Primary Blood Cells

Gene editing using CRISPR systems has gained traction for its potential to treat various diseases, but delivery remains a major challenge. We have previously reported an entirely synthetic gold nanoparticle formulation which can safely and effectively deliver multiple different CRISPR systems as ribonucleoprotein (RNP) into hematopoietic stem cells and immune cells. Here, we describe a modified version of this nanoparticle to simultaneously deliver CRISPR Cas9 RNP and transgene-encoding DNA templates (HDT) as long as ~2.1 kb. We evaluate this HDT-CRISPR-AuNP for gene editing at two loci of interest with different transgene cargo. These nanoparticles successfully deliver gene editing into primary human T cells and hematopoietic stem and progenitor cells with insertion of an antigen-specific T cell receptor transgene. This proof-of-concept immune engineering study demonstrates a simple, synthetic nanoformulation for co-delivery of all cargo required for CRISPR-mediated delivery of transgene cassettes with potential for efficacy in vivo.