No abstract

Efforts to improve CRISPR-Cas9 genome editing systems for lower off-target effects are mostly at the cost of its robust on-target efficiency. To enhance both accuracy and efficiency, we created chimeric SpyCas9 proteins fused with the 5′-to-3′ exonuclease Recombination J (RecJ) or with GFP and demonstrated that transfection of the pre-assembled ribonucleoprotein of the two chimeric proteins into human or plant cells resulted in greater targeted mutagenesis efficiency up to 600% without noticeable increase in off-target effects. Improved activity of the two fusion proteins should enable editing of the previously hard-to-edit genes and thus readily obtaining the cells with designer traits.

No abstract

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.

No abstract