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A Nanobody Toolbox for Recognizing Distinct Epitopes on Cas9.
Jack Boylan1, Rebecca A Shrem1, Isabel C Vallecillo-Viejo2
1Departments of Pharmacology, Vanderbilt University, Nashville, TN 37232, United States.
Journal of Molecular Biology
|October 31, 2024
Summary
Researchers developed nanobodies targeting Streptococcus pyogenes Cas9, a key tool in genetic engineering. These nanobodies bind Cas9 effectively without inhibiting its DNA editing function, offering valuable reagents for scientific research.
Area of Science:
- Molecular Biology
- Biotechnology
- Immunology
Background:
- Cas9 enzymes and their fusions are essential for genetic manipulation.
- Variants like single-base editors and catalytically dead Cas9 (dCas9) expand genome editing capabilities.
Purpose of the Study:
- To generate and characterize a panel of nanobodies against Streptococcus pyogenes Cas9.
- To provide novel reagents for Cas9 research and applications.
Main Methods:
- Immunization of an alpaca with Cas9 to generate a nanobody library.
- Screening and identification of potent Cas9-binding nanobodies.
- Biochemical characterization including ELISA, epitope binning, and mass photometry.
Main Results:
- A panel of nanobodies targeting three distinct epitopes on Cas9 was generated.
- The most effective nanobodies bind both Cas9 and RNA-bound Cas9.
- These nanobodies do not inhibit Cas9's DNA cleavage activity.
- Epitope mapping confirmed non-overlapping binding sites.
Conclusions:
- Novel, non-inhibitory nanobodies against Streptococcus pyogenes Cas9 have been developed.
- These nanobodies recognize Cas9 in different functional states.
- The provided sequences and data facilitate the use of these reagents in the broader scientific community.
