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

Nguyen DN, Roth TL, Li PJ, Chen PA, Apathy R, Mamedov MR, Vo LT, Tobin VR, Goodman D, Shifrut E, Bluestone JA, Puck JM, Szoka FC, Marson A.
Source: Nat Biotechnol
Publication Date: (2019)
Issue: 1: 1
Research Area:
Immunotherapy / Hematology
Regenerative medicine
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
CD4+, human
Species: human
Tissue Origin: blood
CD8+, human
Species: human
Tissue Origin: blood
Culture Media:
96-well Shuttle™ System

HDR templates were mixed and incubated with RNPs for at least 5min prior to mixing with and electroporating into cells. Immediately prior to electroporation in a 96-well format 4D-Nucleofector (Lonza), cells were centrifuged for 10min at 90 g, medium was aspirated, and cells were resuspended in the electroporation buffer P3 (Lonza) using 17–20 µl buffer per 0.5–1.0 × 106 cells. T cells, NK cells, and B cells were electroporated with pulse code EH-115, primary HSPCs with pulse code ER-100, and iPS-derived CD34 HSPCs with pulse code EY-100. Immediately after electroporation, cells were rescued with the addition of 80µL of growth medium directly into the electroporation well, incubated for 10–20min, then removed and diluted to 0.5–1.0 × 106  cells ml-1 in growth medium. Additional fresh growth medium and cytokines were added every 48h.


Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1-5. 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 (tCTSs) 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 approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, 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 approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3+) T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), ?d T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs).