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.

中文翻译：

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化脓性链球菌SpyCas9蛋白单克隆抗体的生产和评估

可编程的DNA裂解酶的生物技术应用被称为聚簇的，规则间隔的短回文重复序列/ CRISPR相关蛋白9（CRISPR / Cas9），继续从农业性状发展到治疗基因治疗。从化脓性链球菌（SpyCas9）分离的Cas9 CRISPR系统被归为II类，由于其高效和鲁棒性而广受欢迎。特异性检测SpyCas9的抗体可以扩大该酶的适用性。在这里，我们报告针对SpyCas9的单克隆抗体的发展。选择四个杂交瘤细胞以表达抗SpyCas9。杂交瘤上清液含有针对SpyCas9的反应性和高度特异性抗体。纯化了抗SpyCas9抗体，并且对其他六个细菌Cas9蛋白没有选择性。可以在48小时内以减少的量在HEK293T细胞中检测到SpyCas9蛋白，表明该抗体可以用于检测经过CRISPR / SpyCas9系统细胞处理后剩余的SpyCas9残留水平。抗SpyCas9抗体可帮助研究人员促进Cas9的进一步研究，以使用诸如酶联免疫吸附测定（ELISA），蛋白质印迹，免疫沉淀和免疫组化染色等技术。