Work

A Nanobody Toolbox for Recognizing Distinct Epitopes on Cas9

Year: 2024

Type: article

Abstract: 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.

Source: Journal of Molecular Biology

DOI: 10.1016/j.jmb.2024.168836

Authors: Jack Boylan, Rebecca A Shrem, Isabel C. Vallecillo-Viejo, Craig L. Duvall, Brian E. Wadzinski, Benjamin W. Spiller

Institutions: Vanderbilt University, National Institutes of Health, National Institute of Allergy and Infectious Diseases

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,420

Funders: Vanderbilt University, School of Medicine, Vanderbilt University, National Heart, Lung, and Blood Institute, National Cancer Institute