Production and assessment of monoclonal antibodies against the SpyCas9 protein ofStreptococcus pyogenes

The biotechnological applications of the programmable DNA-cleaving enzymes known as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) continue to expand from agricultural trait development to therapeutic gene therapies. The Cas9 CRISPR system isolated from Streptococcus pyogenes (SpyCas9) is classified as class II, which is popular due to high efficiency and robustness. Antibodies that specifically detect SpyCas9 could expand the applicability of this enzyme. Here, we report on the development of monoclonal antibodies against SpyCas9. Four hybridoma cells were selected for their expression of anti-SpyCas9. Hybridoma supernatant contained reactive and highly specific antibodies to SpyCas9. Anti-SpyCas9 antibody was purified and was non-selective against six other bacterial Cas9 proteins. SpyCas9 protein could be detected in HEK293T cells in decreasing amounts over a 48 hour period, indicating the antibody could be used to detect residual levels of SpyCas9 remaining following cell treatment with a CRISPR/SpyCas9 system. The anti-SpyCas9 antibody may help researchers facilitate Cas9 for further study for the use of techniques such as enzyme-linked immunosorbent assays (ELISAs), western blot, immunoprecipitation, and immunohistochemical staining.