Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency

Versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies(1–5). Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTS) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency ~2–4 fold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with poly(glutamic acid) improves editing efficiency an additional ~2-fold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses yielding ~2–6 times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), γ-T cells, B cells, NK cells, and primary and iPS-derived(6) hematopoietic stem progenitor cells (HSPCs).