SaCas9, AsCas12a, and CasΦ exhibit comparable pre-existing immunogenicity in naïve individuals
Background
Gene-editing tools like CRISPR-Cas9 hold immense therapeutic potential, but their clinical application is hampered by the immunogenicity of Cas proteins. These proteins, often derived from human pathogens like Staphylococcus aureus, can trigger immune responses, leading to reduced efficacy and safety concerns. Researchers are exploring Cas orthologs from organisms not typically associated with human infections, such as Acidaminococcus sp. or bacteriophages, hoping these alternatives might present a lower immunogenic risk and improve the safety profile of gene therapies.
Study Design
Researchers investigated the immunogenicity risk of three Cas proteins: SaCas9 (from Staphylococcus aureus), AsCas12a (from Acidaminococcus sp.), and CasΦ (from a bacteriophage, Biggiephage). They performed ex vivo and in vitro analyses using samples from unimmunized individuals to assess pre-existing antibody and T cell recognition. Additionally, mass-spectrometry was employed to identify MHC-I-bound peptides for each Cas protein, determining their presentation on common MHC-I proteins found in the North American population.
Results
The study revealed that SaCas9, AsCas12a, and CasΦ were recognized similarly by both antibodies and T cells from unimmunized individuals. This indicates that Cas orthologs from non-pathogenic or phage sources do not inherently offer a less immunogenic profile compared to pathogen-derived Cas9. Mass-spectrometry analysis further identified that peptides from all three Cas proteins—SaCas9, AsCas12a, and CasΦ—were presented on 9 MHC-I proteins commonly found within the North American population. This widespread MHC-I presentation suggests a broad potential for T cell recognition across diverse genetic backgrounds. The findings collectively challenge the assumption that simply sourcing Cas proteins from non-human pathogens or phages would mitigate pre-existing immunogenicity.
SaCas9, AsCas12a, and CasΦ were recognized similarly by antibodies and T cells from unimmunized individuals.
Key Findings
- SaCas9, AsCas12a, and CasΦ were recognized similarly by antibodies from unimmunized individuals.
- T cell responses to SaCas9, AsCas12a, and CasΦ were comparable in unimmunized individuals.
- Peptides from all three Cas proteins were presented on 9 MHC-I proteins commonly found in North America.
- AsCas12a and CasΦ do not present a less immunogenic alternative to SaCas9 based on pre-existing immunity.
Why It Matters
This research significantly impacts the development of gene-editing therapies by highlighting that simply choosing Cas proteins from non-pathogenic sources does not guarantee reduced immunogenicity. Systematic immunogenicity evaluation is critical for all Cas proteins intended for clinical use, regardless of their origin. For biohackers and researchers, this means that the choice of Cas ortholog for gene-editing applications should not solely rely on the source organism's pathogenicity. Instead, thorough pre-screening for immune responses against specific Cas proteins is essential to predict and potentially mitigate adverse immune reactions in human subjects, pushing the field towards more rigorous pre-clinical assessment before clinical translation.
cas9
cas12a
casphi
gene-editing
immunogenicity
crispr