CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays

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

“…We successfully recapitulated the known DYKxxDxx binding motif of the M2 antibody ( Osada et al., 2009 ; Srila and Yamabhai, 2013 ), demonstrating the capability of CasPlay to identify amino acid residues that are critical to coordinate antibody binding ( Figure 2 C). The barcode enrichment analysis performed comparably to PICASSO experiments using the same dCas9-displayed FLAG variant library with M2 and a fluorescence-based microarray scanning assay ( Barber et al., 2021 ) (R 2 = 0.79; Figure 2 D), showing the ability of NGS-based gRNA sequencing to identify antibody-bound peptides.

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.

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“…The DNA library is then cloned in a single pool into an expression vector for the E . coli -based production of the peptide library as C-terminal fusions to dCas9 bound to a gRNA barcode that serves as a unique identifier for the peptide ( Figure S1 ) ( Barber et al., 2021 ). To facilitate sequencing of gRNAs for CasPlay, we added a 20 nt universal sequence to the 5′ end of the gRNAs to permit amplification by RT-PCR.…”

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

“…dCas9 fused Peptide libraries are barcoded with unique sgRNA, multiplexed for viral epitope mapping, and can be customized for rapid large scale protein studies. This platform, which was referred to as PICASSO, can locate user-programmed sites on a microarray surface containing DNA sequences complementary to each peptide’s sgRNA from a single mixed pool of peptides fused with dCas9 [ 6 ]. The design of the assays includes the co-expression of dCas9 fusion peptides and uniquely designed sgRNA on the same plasmid so that each E. coli produces a pair of sgRNA and dCas9 fusion peptides, which can be isolated from a single mixed culture of E.coli .…”

“…Hence, a large number of complex peptide libraries can be self-assembled on a microarray chip and the screening of complex peptides can occur at the same time. After applying a dCas9-sgRNA library to a dsDNA microarray, PICASSO permits the screening of mutagenesis peptide libraries in a couple of hours with accuracy, eliminating the requirement of Next-generation sequencing [ 6 ].…”

“…Though the majority of the literature is focused on the detection of NA by CRISPR-based systems, recent studies indicate that CRISPR-based systems also have the potential to diagnose using elements other than NA targets (proteins, antibiotics, monatomic ions and metabolites of amino acids, etc.) [ 2 , 6 , 7 ]. The present review was conceptualized to discuss the developments of CRISPR/Cas-based detection strategies utilizing different Cas subsystems, provide an overview, and investigate the mechanism of repurposing CRISPR/Cas for gene editing to the detection of biomolecules.…”