Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Endotoxin contamination in commercially available Cas9 proteins potentially induces T-cell mediated responses

Gene Therapy volume 30, pages 575–580 (2023)Cite this article

Subjects

Abstract

Immune responses to Cas proteins have been demonstrated recently and these may prove to be an impediment to their clinical use in gene editing. To make meaningful assessments of Cas9 immunogenicity during drug development and licensure it is imperative the reagents are free of impurities that could affect in vitro assessments of immunogenicity. Here we address the issue of endotoxin levels in laboratory grade Cas9 proteins used to measure T-cell memory responses. Many of these reagents have not been developed for immunogenicity assays, are or microbial origin and carry varying levels of endotoxin. The use of these reagents, off the shelf, without measuring endotoxin levels is likely to introduce incorrect estimates of the prevalence of memory T-cell responses in research studies. We demonstrate wide variation in endotoxin levels in Cas9 proteins from seven suppliers. Different lots from the same supplier also contained varying levels of endotoxin. ELISPOT assays showed similar large variations in the interferon-γ signals. Finally, when we carried out endotoxin depletion in four Cas9 proteins with strong signals in the ELISPOT assay, we found dampening of the interferon-γ signals.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Endotoxin content in Cas9 proteins sourced from different suppliers.
Fig. 2: Dose-response curves for IFN-γ release in an ELISPOT assay using increasing concentrations of LPS.
Fig. 3: IFN-γ release in an ELISPOT assay using Cas9 proteins from different suppliers.
Fig. 4: Association between endotoxin contamination and IFN-γ release in an ELISPOT assay for Cas9 proteins from different suppliers.
Fig. 5: Removal of endotoxin from Cas9 proteins reduces IFN-γ release.

Similar content being viewed by others

References

  1. Gaj T, Sirk SJ, Shui SL, Liu J. Genome-editing technologies: principles and applications. Cold Spring Harb Perspect Biol. 2016;8:a023754.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Li H, Yang Y, Hong W, Huang M, Wu M, Zhao X. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduct Target Ther. 2020;5:1.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Pickar-Oliver A, Gersbach CA. The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol. 2019;20:490–507.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Rodriguez-Rodriguez DR, Ramirez-Solis R, Garza-Elizondo MA, Garza-Rodriguez ML, Barrera-Saldana HA. Genome editing: A perspective on the application of CRISPR/Cas9 to study human diseases (Review). Int J Mol Med. 2019;43:1559–74.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Chen H, Shi M, Gilam A, Zheng Q, Zhang Y, Afrikanova I, et al. Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII. Sci Rep. 2019;9:16838.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat Methods. 2016;13:868–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520:186–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Stone D, Long KR, Loprieno MA, De Silva Feelixge HS, Kenkel EJ, Liley RM, et al. CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice. Mol Ther Methods Clin Dev. 2021;20:258–75.

    Article  CAS  PubMed  Google Scholar 

  9. Charlesworth CT, Deshpande PS, Dever DP, Camarena J, Lemgart VT, Cromer MK, et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med. 2019;25:249–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Simhadri VL, McGill J, McMahon S, Wang J, Jiang H, Sauna ZE. Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population. Mol Ther Methods Clin Dev. 2018;10:105–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wagner DL, Amini L, Wendering DJ, Burkhardt LM, Akyuz L, Reinke P, et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat Med. 2019;25:242–8.

    Article  CAS  PubMed  Google Scholar 

  12. Food and Drug Administration. Immunogenicity Assessment for Therapeutic Protein Products (Guidance for Industry) 2014.

  13. Rosenberg AS, Sauna ZE. Immunogenicity assessment during the development of protein therapeutics. J Pharm Pharmacol. 2018;70:584–94.

    Article  CAS  PubMed  Google Scholar 

  14. Fessenden M. Technologies to watch in 2019. Nature. 2019;565:521–3.

    Article  CAS  PubMed  Google Scholar 

  15. Alexander C, Rietschel ET. Bacterial lipopolysaccharides and innate immunity. J Endotoxin Res. 2001;7:167–202.

    CAS  PubMed  Google Scholar 

  16. Triantafilou M, Triantafilou K. Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends Immunol. 2002;23:301–4.

    Article  CAS  PubMed  Google Scholar 

  17. Kumar H, Kawai T, Akira S. Pathogen recognition by the innate immune system. Int Rev Immunol. 2011;30:16–34.

    Article  CAS  PubMed  Google Scholar 

  18. Rossol M, Heine H, Meusch U, Quandt D, Klein C, Sweet MJ, et al. LPS-induced cytokine production in human monocytes and macrophages. Crit Rev Immunol. 2011;31:379–446.

    Article  CAS  PubMed  Google Scholar 

  19. Schwarz H, Gornicec J, Neuper T, Parigiani MA, Wallner M, Duschl A, et al. Biological Activity of Masked Endotoxin. Sci Rep. 2017;7:44750.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ferdosi SR, Ewaisha R, Moghadam F, Krishna S, Park JG, Ebrahimkhani MR, et al. Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes. Nat Commun. 2019;10:1842.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Rosenberg AS, Worobec, A. A Risk-Based Approach to Immunogenicity Concerns of Therapeutic Protein Products, Part 2: Considering Host-Specific and Product-Specific Factors Impacting Immunogenicity. BioPharm International. 2004;17:12.

    Google Scholar 

  22. Budwell G. Uh-Oh! CRISPR Gene-Editing Stocks May Be Worthless. 2018.

  23. Daley J. Immunity May Make CRISPR-Based Therapies Ineffective. TheScientist. 2018.

  24. Ledford H. How the immune system could stymie some CRISPR gene therapies. Nature. 2018.

  25. Zhang S. You May Already Be Immune to CRISPR. 2018.

  26. Tennenberg SD, Weller JJ. Endotoxin activates T cell interferon-gamma secretion in the presence of endothelium. J Surg Res. 1996;63:73–6.

    Article  CAS  PubMed  Google Scholar 

  27. Dawson M. ENDOTOXIN LIMITS for Parenteral Drug Products. BET White Paper. 2017;1:2.

    Google Scholar 

  28. Food and Drug Administration. Pyrogen and Endotoxins Testing: Questions and Answers (Guidance for Industry). 2012.

  29. The United States Pharmacopeial Convention. Bacterial Endotoxin Test. Second Supplement to USP 35–NF 30. 2012.

  30. Schoenborn JR, Wilson CB. Regulation of interferon-gamma during innate and adaptive immune responses. Adv Immunol. 2007;96:41–101.

    Article  CAS  PubMed  Google Scholar 

  31. Rock KL, Reits E, Neefjes J. Present Yourself! By MHC Class I and MHC Class II Molecules. Trends Immunol. 2016;37:724–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Weenink SM, Gautam AM. Antigen presentation by MHC class II molecules. Immunol Cell Biol. 1997;75:69–81.

    Article  CAS  PubMed  Google Scholar 

  33. La Gruta NL, Gras S, Daley SR, Thomas PG, Rossjohn J. Understanding the drivers of MHC restriction of T cell receptors. Nat Rev Immunol. 2018;18:467–78.

    Article  PubMed  Google Scholar 

  34. Ratanji KD, Dearman RJ, Kimber I, Thorpe R, Wadhwa M, Derrick JP. Editor’s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response. Toxicol Sci. 2016;153:258–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Endotoxin-free protein production-ClearColiTM technology. Nature Methods. 2013;10:916.

  36. Varma TK, Lin CY, Toliver-Kinsky TE, Sherwood ER. Endotoxin-induced gamma interferon production: contributing cell types and key regulatory factors. Clin Diagn Lab Immunol. 2002;9:530–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Alagoz M, Kherad N. Advance genome editing technologies in the treatment of human diseases: CRISPR therapy (Review). Int J Mol Med. 2020;46:521–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Cai L, Fisher AL, Huang H, Xie Z. CRISPR-mediated genome editing and human diseases. Genes Dis. 2016;3:244–51.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

ZES is funded by intramural grants from the US Food and Drug Administration. Custom manufactured, endotoxin free products, CON-SaCas9 and CON-SpCas9 were a kind gift from Editas Medicine.

Author information

Authors and Affiliations

  1. Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA

    Vijaya L. Simhadri, Joseph R. McGill & Zuben E. Sauna

Authors
  1. Vijaya L. Simhadri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joseph R. McGill
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zuben E. Sauna
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

ZES, conceptualised the study. VLS, performed experiments. ZES, VLS, JRM, designed experiments and analysed data. ZES, VLS, JRM, wrote the paper. ZES obtained funding.

Corresponding author

Correspondence to Zuben E. Sauna.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Simhadri, V.L., McGill, J.R. & Sauna, Z.E. Endotoxin contamination in commercially available Cas9 proteins potentially induces T-cell mediated responses. Gene Ther 30, 575–580 (2023). https://doi.org/10.1038/s41434-021-00301-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41434-021-00301-6

Search

Advanced search

Quick links