Departments of Pharmacology, Vanderbilt University, Nashville, TN 37232, United States.
National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, United States; Departments of Biomedical Engineering, Vanderbilt University, Nashville, TN 37232, United States.
J Mol Biol. 2024 Dec 1;436(23):168836. doi: 10.1016/j.jmb.2024.168836. Epub 2024 Oct 30.
Cas9s and fusions of Cas9s have emerged as powerful tools for genetic manipulations. Fusions of Cas9 with other DNA editing enzymes have led to variants capable of single base editing and catalytically dead Cas9s have emerged as tools to specifically target desired regions of a genome. Here we describe the generation of a panel of nanobodies directed against three unique epitopes on Streptococcus pyogenes Cas9. The nanobodies were identified from a nanobody library derived from an alpaca that had been immunized with Cas9. The most potent binders recognize Cas9 and RNA bound Cas9 equally well and do not inhibit Cas9 cleavage of target DNA. These nanobodies bind non-overlapping epitopes as determined by ELISA based epitope binning experiments and mass photometry. We present the sequences of these clones and supporting biochemical data so the broader scientific community can access these reagents.
Cas9 及其融合蛋白已成为基因操作的有力工具。Cas9 与其他 DNA 编辑酶的融合产生了能够进行单碱基编辑的变体，而催化失活的 Cas9 则成为特异性靶向基因组特定区域的工具。在这里，我们描述了一组针对化脓性链球菌 Cas9 上三个独特表位的纳米抗体的产生。这些纳米抗体是从用 Cas9 免疫的羊驼的纳米抗体文库中鉴定出来的。最有效的结合物能够同等程度地识别 Cas9 和与 RNA 结合的 Cas9，并且不会抑制 Cas 9 对靶 DNA 的切割。这些纳米抗体通过基于 ELISA 的表位分组实验和质量光度法确定了非重叠的表位。我们呈现了这些克隆的序列和支持的生化数据，以便更广泛的科学界可以使用这些试剂。
