Pre-existing antibody and T cell responses to SaCas9, AsCas12a and CasΦ are comparable in naïve individuals.

Cas9 proteins are derived from human pathogens and are immunogenic, thereby raising potential safety concerns and limiting clinical effectiveness when using Cas9 as a gene-editing tool. Cas orthologs developed from organisms not directly associated with human infections may thus be safer. Here we compare the immunogenicity risk of SaCas9 (derived from the human pathogen, Staphylococcus aureus), AsCas12a (derived from the human commensal, Acidaminococcus sp.) and CasΦ (derived from a bacteria phage, Biggiephage). Ex vivo and in vitro analyses show that SaCas9, AsCas12a and CasΦ are recognized similarly by antibodies and T cells from unimmunized individuals. Using mass-spectrometry to identify MHC-I-bound peptides, we find SaCas9, AsCas12a and CasΦ peptides presented on 9 MHC-I proteins commonly found in the North American population. Our results thus indicate that AsCas12a and CasΦ do not present a less immunogenic alternative to Cas9, and underscore the need for systematic immunogenicity evaluation of all Cas proteins intended for clinical use.