CasPlay provides a gRNA-barcoded CRISPR-based display platform for antibody repertoire profiling

Abstract

Protein display technologies link proteins to distinct nucleic acid sequences (barcodes), enabling multiplexed protein assays via DNA sequencing. Here, we develop Cas9 display (CasPlay) to interrogate customized peptide libraries fused to catalytically inactive Cas9 (dCas9) by sequencing the guide RNA (gRNA) barcodes associated with each peptide. We first confirm the ability of CasPlay to characterize antibody epitopes by recovering a known binding motif for a monoclonal anti-FLAG antibody. We then use a CasPlay library tiling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteome to evaluate vaccine-induced antibody reactivities. Using a peptide library representing the human virome, we demonstrate the ability of CasPlay to identify epitopes across many viruses from microliters of patient serum. Our results suggest that CasPlay is a viable strategy for customized protein interaction studies from highly complex libraries and could provide an alternative to phage display technologies.

Keywords: CRISPR; Cas9; antibody binding; biotechnology; peptide display; protein display.

Graphical abstract

Figure 1

CasPlay library design and workflow Customized peptide libraries are encoded in an oligonucleotide library, with each peptide sequence paired with a unique 20 bp nucleic acid sequence to be used as the gRNA barcode. The peptides are expressed in E. coli as fusions to dCas9 bound to unique gRNA barcode sequences and purified in a single batch. The gRNA sequences have a universal 5′ constant region to facilitate amplification by RT-PCR. This library is then used for immunoprecipitation experiments using human serum antibodies. Peptides bound to these antibodies are identified by nucleotide sequencing of the enriched gRNA barcodes.

Figure 2

CasPlay experiments to map an antibody epitope with single amino acid resolution (A) A FLAG peptide (DYKDDDK) saturation mutagenesis library was produced for CasPlay in which every possible single amino acid substitution was performed along the length of the FLAG epitope, and each variant peptide was associated with a unique gRNA barcode. (B) Sequencing-based enrichment analysis of gRNA barcodes revealed critical amino acid positions to coordinate M2 antibody binding to the FLAG peptide epitope. Enrichment data averaged across 4 gRNA barcode replicates and two independent experimental replicates. (C) Enrichment of the DYKxxDxx motif for antibody binding was identified by sequence logo (Schneider and Stephens, 1990; Tareen and Kinney, 2020), consistent with previous studies (Layton et al., 2019). (D) Sequence enrichment of FLAG variant peptides by CasPlay performed comparably with PICASSO, the microarray-based strategy for studying custom dCas9-fusion peptide libraries with fluorescence imaging readout (Barber et al., 2021).

Figure 3

SARS-CoV-2 epitope mapping by CasPlay (A) A peptide library tiling the SARS-CoV-2 proteome (excluding ORF1ab) from a previous study (Barber et al., 2021) was presented by CasPlay. (B) CasPlay experiments were performed using serum samples from 8 samples collected prior to December 2020 (“pre-COVID”), 8 patients following COVID-19 infection (“convalescent”), 8 individuals prior to receiving a vaccine, and the same 8 individuals between 2 weeks and 3 months after receiving the second dose of an mRNA vaccine. gRNA barcode enrichment analysis by CasPlay revealed epitope regions in the spike and/or nucleocapsid proteins recognized by patient antibodies in convalescent and vaccinated patient samples. Enrichment is calculated as normalized output gRNA sequencing reads divided by pre-enriched normalized sequencing reads, with data averaged across 4 gRNA barcode replicates and two independent experimental replicates (see STAR methods for further details).

Figure 4

CasPlay enables interrogation of antibodies on human virome scale (A) 122,501-member tiled peptide-based representation of the human virome displayed by CasPlay based on peptide libraries from previous studies (Shrock et al., 2020; Xu et al., 2015). Each peptide was paired with an orthogonal gRNA barcode, with two separate barcodes assigned to each peptide (245,002 library members total). Epitope tags recognized by commercial monoclonal antibodies (e.g., FLAG, HA, myc) were also encoded as control peptides within the library. (B) CasPlay control experiments using anti-FLAG, anti-HA, and anti-myc antibodies identified all ten corresponding control replicates and none of the off-target epitope tag peptides, within the context of the entire 245,002-member library. Error bars show SD centered at the mean. (C) CasPlay was able to identify subregions of viral proteins commonly targeted by multiple patients (public epitopes) from within the 245,002-member library. Examples shown from adenovirus A, Epstein-Barr virus, and RSV. Z scores were averaged for gRNA barcode duplicates across two independent patient samples.

Figure 5

CasPlay is compatible with full-length protein display and applications (A) Synthetic antibodies were expressed as C-terminal fusions to dCas9 with unique gRNA barcodes. A nanobody recognizing a peptide from β-catenin (Braun et al., 2016; Traenkle et al., 2015) and an scFv recognizing the spike protein from SARS-CoV-2 (Wang et al., 2021) were paired with unique gRNAs. (B) Target antigens of the synthetic antibodies were adsorbed to a plate surface and then incubated with the dCas9-antibody fusions. Primers specific to the gRNA sequences were then used to RT-PCR the gRNAs associated with each antibody to detect binding of the antibody to its target antigen. (C) Densitometry of RT-PCR amplicons on an agarose gel reveal specific retention and detection of each synthetic antibody only in the presence of its respective analyte, indicating synthetic antibody functionality. n = 3 independent replicates for each condition. Mean with SD plotted.