Efficient intracellular delivery of CRISPR-Cas9 ribonucleoproteins using dendrimer nanoparticles for robust genomic editing

CRISPR-Cas9, a flexible and efficient genome editing technology, is currently limited by the challenge of delivering the large ribonucleoprotein complex intracellularly and into the nucleus. Existing delivery techniques/vectors are limited by their toxicity, immunogenicity, scalability, and lack of specific cell-targeting ability. This study presents a neutral, non-toxic dendrimer conjugate construct that shows promise in overcoming these limitations. We covalently-conjugated S. pyogenes Cas9–2NLS (Cas9-nuclear localization sequence) endonuclease to a generation-6 hydroxyl PAMAM dendrimer through a glutathione-sensitive disulfide linker via highly specific inverse Diels-alder click reaction (IEDDA), and a single guide RNA (sgRNA) was complexed to the Cas9-dendrimer conjugate nano-construct (D-Cas9). D-Cas9- RNP produces robust genomic deletion in vitro of GFP in HEK293 cells (∼100 %) and VEGF in a human pigmental epithelium cell line (ARPE-19) (20 %). The uptake of the D-Cas9-RNP constructs on similar timescales as small molecules highlights the robustness of the biophysical mechanisms enabling the dendrimer to deliver payloads as large as Cas9, while retaining payload functionality. This promising conjugation approach enabled better stability to the neutral construct. Combined with recent advances in hydroxyl dendrimer delivery technologies in the clinic, this approach may lead to advances in ‘neutral’ dendrimer-enabled non-toxic, cell-specific, highly efficient in vitro and in vivo genome editing.