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


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.

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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.


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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.

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

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Correspondence to Zuben E. Sauna.

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Simhadri, V.L., McGill, J.R. & Sauna, Z.E. Endotoxin contamination in commercially available Cas9 proteins potentially induces T-cell mediated responses. Gene Ther (2021).

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