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Conserved Binding Sites

Conserved Binding Sites

Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...

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Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

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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.

Keywords:

Cas9 Epitope bin nanobody

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Published on: March 31, 2022

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.