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

  • Nat Biotechnol. 2020 Jan;38(1):44-49. doi: 10.1038/s41587-019-0325-6.
David N Nguyen  #  1  2  3  4 ,  Theodore L Roth  #  2  3  4  5  6 ,  P Jonathan Li  2  3  4 ,  Peixin Amy Chen  2  3  4 ,  Ryan Apathy  2  3  4 ,  Murad R Mamedov  2  3  4 ,  Linda T Vo  3 ,  Victoria R Tobin  2  3  4 ,  Daniel Goodman  2  3  4 ,  Eric Shifrut  2  3  4 ,  Jeffrey A Bluestone  3  7 ,  Jennifer M Puck  8 ,  Francis C Szoka  9 ,  Alexander Marson  10  11  12  13  14  15  16
Affiliations
  • 1. Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
  • 2. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
  • 3. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA.
  • 4. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
  • 5. Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.
  • 6. Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
  • 7. Sean N. Parker Autoimmune Research Laboratory, University of California, San Francisco, San Francisco, CA, USA.
  • 8. Division of Allergy, Immunology, and Bone Marrow Transplantation, Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA.
  • 9. Department of Bioengineering and Therapeutic Sciences, School of Pharmacy, University of California, San Francisco, San Francisco, CA, USA.
  • 10. Department of Medicine, University of California, San Francisco, San Francisco, CA, USA. [email protected].
  • 11. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. [email protected].
  • 12. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. [email protected].
  • 13. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. [email protected].
  • 14. Chan Zuckerberg Biohub, San Francisco, CA, USA. [email protected].
  • 15. UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. [email protected].
  • 16. Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA. [email protected].
  • # Contributed equally.
PMID: 31819258 DOI: 10.1038/s41587-019-0325-6
Abstract

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), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs).