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


Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1,2,3,4,5. 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).

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Fig. 1: Truncated tCTSs in HDR templates increase large non-viral knock-in efficiency.
Fig. 2: Stabilizing Cas9 RNP nanoparticles with anionic polymers improves editing outcomes.
Fig. 3: PGA-stabilized Cas9 RNP and tCTS-modified HDR templates improved knock-in gene editing outcomes across a variety of genetic loci and clinically relevant immune cell types.

Data availability

Amplicon sequencing data have been deposited in the National Institutes of Health NCBI SRA (Bioproject PRJNA564604), and flow cytometry raw data files are available upon request. Plasmids containing the HDR template sequences used in the study are available through AddGene (Supplementary Table 1), and annotated DNA sequences for all constructs are available upon request.


  1. 1.

    Yin, H., Xue, W. & Anderson, D. G. CRISPR–Cas: a tool for cancer research and therapeutics. Nat. Rev. Clin. Oncol. 16, 281––295 (2019).

    Article  Google Scholar 

  2. 2.

    Dunbar, C. E. et al. Gene therapy comes of age. Science 359, eaan4672 (2018).

    Article  Google Scholar 

  3. 3.

    Cornu, T. I., Mussolino, C. & Cathomen, T. Refining strategies to translate genome editing to the clinic. Nat. Med. 23, 415––423 (2017).

    Article  Google Scholar 

  4. 4.

    David, R. M. & Doherty, A. T. Viral vectors: the road to reducing genotoxicity. Toxicol. Sci. 155, 315––325 (2017).

    Article  Google Scholar 

  5. 5.

    Roth, T. L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405––409 (2018).

    Article  Google Scholar 

  6. 6.

    Vo, L. T. et al. Regulation of embryonic haematopoietic multipotency by EZH1. Nature 553, 506––510 (2018).

    Article  Google Scholar 

  7. 7.

    Pouton, C. W., Wagstaff, K. M., Roth, D. M., Moseley, G. W. & Jans, D. A. Targeted delivery to the nucleus. Adv. Drug Deliv. Rev. 59, 698––717 (2007).

    Article  Google Scholar 

  8. 8.

    Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014).

    Article  Google Scholar 

  9. 9.

    Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17, 5––15 (2016).

    Article  Google Scholar 

  10. 10.

    Jiang, F. & Doudna, J. A. CRISPR-Cas9 Structures and Mechanisms. Annu. Rev. Biophys. 46, 505––529 (2017).

    Article  Google Scholar 

  11. 11.

    Luecke, S. et al. cGAS is activated by DNA in a length-dependent manner. EMBO Rep. 18, 1707––1715 (2017).

    Article  Google Scholar 

  12. 12.

    Richardson, C. D., Ray, G. J., Bray, N. L. & Corn, J. E. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes. Nat. Commun. 7, 12463 (2016).

    CAS  Article  Google Scholar 

  13. 13.

    Bernkop-Schnurch, A. Strategies to overcome the polycation dilemma in drug delivery. Adv. Drug Deliv. Rev. 136-137, 62––72 (2018).

    Article  Google Scholar 

  14. 14.

    Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216––1224 (2018).

    Article  Google Scholar 

  15. 15.

    Tsai, S. Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat. Methods 14, 607––614 (2017).

    Article  Google Scholar 

  16. 16.

    Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224––226 (2019).

    Article  Google Scholar 

  17. 17.

    Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380––1389 (2013).

    Article  Google Scholar 

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We thank members of the Marson laboratory, C. Jeans and the QB3 MacroLab, and A. Dolor for suggestions and technical assistance. This research was supported by NIH grants DP3DK111914-01, R01DK1199979, and P50GM082250 (A.M.), a grant from the Keck Foundation (A.M.), gifts from Jake Aronov, Barbara Bakar, and the American Endowment Foundation (A.M.), a gift from the UCSF Diabetes Center, a gift from the Jeffrey Modell Foundation, and a National Multiple Sclerosis Society grant (A.M., CA 1074-A-21). D.N.N. was supported by the UCSF Biology of Infectious Diseases Training Program (T32A1007641), an NIH Loan Repayment Program grant (L40 AI140341), and the CIS CSL Behring Fellowship Award. T.L.R. was supported by the UCSF Medical Scientist Training Program (T32GM007618), the UCSF Endocrinology Training Grant (T32 DK007418), and the National Institute of Diabetes and Digestive and Kidney Disorders (F30DK120213). L.T.V. is supported by a Damon Runyon Postdoctoral Research Fellowship. A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund, is an investigator at the Chan Zuckerberg Biohub, and has received funding from the Innovative Genomics Institute (IGI) and the Parker Institute for Cancer Immunotherapy. The UCSF Flow Cytometry Core was supported by the Diabetes Research Center grant NIH P30 DK063720.

Author information




D.N.N., T.L.R., and A.M. designed the study. T.L.R. conceived the template ‘shuttle’ system and performed all ‘shuttle’ optimization. D.G. suggested the use of truncated Cas9 target sequences. D.N.N. conceived the polymer stabilization of RNP system and performed all polymer optimizations. D.N.N., T.L.R., P.J.L., P.A.C., R.A., M.R.M., L.T.V., V.R.T., D.G., E.S., J.A.B., J.M.P., and F.C.S. contributed to the design and completion of experiments combining the shuttle and polymer systems in additional primary cell types. D.N.N., T.L.R., and A.M. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Alexander Marson.

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Competing interests

A.M. is a co-founder of Spotlight Therapeutics. T.L.R. and A.M. are co-founders of ArsenalBio. J.A.B. is a founder of Sonoma Biotherapeutics. A.M. serves on the scientific advisory board of PACT Pharma, is an advisor to Trizell, and was a former advisor to Juno Therapeutics. The Marson laboratory has received sponsored research support from Juno Therapeutics, Epinomics, and Sanofi, as well as a gift from Gilead. Patents have been filed based on the findings described here. All other authors have no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figures 1–15 and Supplementary Tables 2 and 3

Reporting Summary

Supplementary Table 1

List of HDR templates, DNA primers, gRNA sequences, and target amplicons used in this study. For sequences please see uploaded MS Excel file.

Supplementary Table 4

Statistics for Figure 1

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Nguyen, D.N., Roth, T.L., Li, P.J. et al. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nat Biotechnol 38, 44–49 (2020).

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