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Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing
Published on: March 15, 2019
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CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling
Karl W Barber1,2, Ellen Shrock1,2, Stephen J Elledge1,2
1Division of Genetics, Brigham and Women's Hospital, Howard Hughes Medical Institute, Boston, MA 02115, USA.
Cell Reports Methods
|October 31, 2022
Summary
We developed Cas9 display (CasPlay), a novel protein display technology. CasPlay uses guide RNA barcodes to identify protein interactions, enabling efficient analysis of complex peptide libraries for epitope mapping and viral diagnostics.
Area of Science:
- Molecular Biology
- Biotechnology
- Immunology
Background:
- Protein display technologies link proteins to nucleic acid barcodes for multiplexed assays.
- Current methods can be limited in scope or require extensive sample volumes.
Purpose of the Study:
- To develop a novel protein display technology, Cas9 display (CasPlay), for interrogating customized peptide libraries.
- To demonstrate CasPlay's utility in characterizing antibody epitopes and evaluating immune responses to viral pathogens.
Main Methods:
- Cas9 display (CasPlay) technology was developed, fusing peptides to catalytically inactive Cas9 (dCas9).
- Guide RNA (gRNA) barcodes associated with each peptide were sequenced to identify protein interactions.
- CasPlay was validated using a known antibody epitope and applied to SARS-CoV-2 and human virome peptide libraries.
Main Results:
- CasPlay successfully identified a known binding motif for a monoclonal anti-FLAG antibody.
- Vaccine-induced antibody reactivities against the SARS-CoV-2 proteome were evaluated using CasPlay.
- CasPlay identified viral epitopes from microliters of patient serum across a diverse human virome library.
Conclusions:
- CasPlay is a viable strategy for customized protein interaction studies using complex libraries.
- This technology offers a potential alternative to existing methods like phage display for epitope mapping and diagnostics.
