Archaeal and bacteriophage derived Cas proteins demonstrate memory T cell response in humans

Abstract

Abstract The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a powerful gene editing tool with clinical applications in the pipeline. In vivodelivery of Cas proteins could induce immune responses which would limit the utility of the technology. Previously, we and others have demonstrated pre-existing immunity to Cas9 proteins derived from Staphylococcus aureus(Sa) and Streptococcus pyogenes(Sp) in human populations. As these Cas proteins are of microbial origin this finding was not surprising. There are numerous naturally occurring alternatives to Cas9 and many of these are being leveraged for gene editing. Alternatives to Cas9 that have successfully been used in gene editing technologies include Cas12a (derived from Acidaminococcus sp), Cas14a (derived from DPANN, a super-phylum of archaea) and Casϕ (derived from a huge bacteriophage). It has been postulated that humans would not have pre-existing immunity to some of these Cas proteins. In the comparative study described here, we evaluated pre-existing antibodies and memory T-cell responses to Cas9, Cas12a, Cas14a and Casϕ from the same 18 healthy donors for all proteins. Pre-existing antibodies were identified using ELISA and memory T-cell responses were measured using the ELISpot assay. All four Cas proteins tested positive for both assays in at least some donors. It has been speculated that pre-existing immunity to Cas9 proteins is due to human exposure to the pathogenic bacteria they are derived from. Consequently, Cas14a and Casϕ should not be associated with pre-existing immunity. However, our results show that anti-Cas14a and anti-Casϕ antibodies were detected in ~50% of the study population and these donors also showed memory T-cell responses.