CRISPR-Based Tech Could Revolutionize Antibody-Based Diagnostics

Lead author, Karl Barber with a PICASSO microarray. [Karl Barber, Schmidt Science Fellows]
Scientists have used an adaptation of the genome editing technology CRISPR to develop a new peptide display platform that can be used as a tool to identify antibodies in patient blood samples. Researchers from the Howard Hughes Medical Institutes and Harvard Medical School suggest the new technology, which they’ve called PICASSO (peptide immobilization by Cas9-mediated self-organization), could inspire a new class of medical diagnostics, and springboard a host of other applications.

For their reported study in Molecular Cell, which is titled, “CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays,” the investigators used the platform to detect antibodies binding to proteins derived from pathogens, including SARS-CoV-2 from the blood of recovering COVID-19 patients. The work was led by Stephen Elledge, PhD, at Harvard Medical School and Brigham and Women’s Hospital. The paper’s first author, Karl Barber, PhD, is a 2018 Schmidt science fellow, with much of the work developing the technology taking place during his fellowship research placement in corresponding author Elledge’s laboratory.

CRISPR-Cas9 technology has been adapted for a range of applications in precise genome editing and transcriptional regulation, which has enabled scientists to effectively manipulate genetic sequences “at will,” the authors noted. But the ability of different Cas nuclease enzymes to recognize and respond to nucleic acid sequences complementary to their bound single-guide RNAs (sgRNAs) has also “further inspired the development of in vitro CRISPR-based technologies,” including rapid point-of-care pathogen identification and nucleic acid-responsive smart hydrogels. “CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection,” they noted.

The PICASSO technology developed by Barber and colleagues uses a modified Cas9 enzyme to facilitate the study of custom peptide libraries and overcome the limitations of current display technologies. “In developing PICASSO, we have demonstrated multiplexed peptide library self-assembly using a CRISPR-based system, making rapid custom protein studies feasible in any laboratory with access to common molecular biology reagents,” they wrote. The PICASSO approach harnesses customizable collections of proteins, which are attached to a catalytically inactive Cas9 enzyme (dCase9). This variant will bind to DNA, but not cut it, as it would for genetic modification applications. The peptide collections are fused to the dCAS9, and barcoded with unique single guide RNA (sgRNA) sequences.

These dCas9-peptide fusions then self-assemble to positions on a DNA microarray surface complementary to their sgRNA barcodes. So when applied to a microchip sporting thousands of unique DNA molecules, each protein within the mixture will self-assemble to the position on the chip containing its corresponding DNA sequence. “… the single mixed pool of dCas9-fusion peptides is able to localize to user-programmed positions on a microarray surface containing DNA sequences complementary to each peptide’s sgRNA barcode,” the team noted. The resulting DNA-templated self-assembling peptide microarrays can then be used for large-scale protein studies. “dCas9-fusion display and self-assembling microarray construction via PICASSO circumvent many of the caveats of other display platforms, making custom peptide library studies faster and more broadly accessible,” the investigators stated.

CRISP SARS-CoV-2 graphical abstract
Source: Schmidt Science Fellows

Describing the technology, Barber said, “Imagine you want to paint a picture on a canvas, but instead of painting in a normal fashion, you mix all of your paints together, splash it on the canvas, and the perfect picture emerges. With our new technique, you place DNA molecules at defined locations on a surface and each protein from a mixture will then self-assemble to its corresponding DNA sequence, like an automated paint-by-number kit. The resulting DNA-templated protein microarrays allow you to quickly identify antibodies in clinical samples that recognize whatever proteins you are interested in.” So, by applying a blood sample to the PICASSO microarray, the proteins on the microchip that are recognized by patient antibodies can be identified.

Barber commented, “In this work, we demonstrated the application of PICASSO for protein studies, creating a tool that we believe could be quickly adapted for medical diagnostics. Our protein self-assembly technique could also be harnessed for the development of new biomaterials and biosensors just by attaching DNA targets to a scaffold and allowing Cas9-linked proteins to bind.” The authors further claimed, “Without next-generation sequencing requirements or multiple rounds of selection, PICASSO can be performed over the course of a couple hours after dCas9-sgRNA library application to a dsDNA microarray (compared with several days or weeks) and avoids sequencing costs and PCR artifacts associated with nucleic-acid-based displays.”

Group leader Elledge, commented: “One of the most exciting aspects of this work is the demonstration of how CRISPR can be applied in an entirely new setting. Previously, CRISPR has been used primarily for gene editing and the detection of DNA or RNA. PICASSO brings the power of CRISPR into a new realm of protein studies, and the molecular self-assembly strategy we show may assist in developing new research and diagnostic tools.”

And in the future, the team noted, “ … CRISPR-based protein display and PICASSO may also be developed for classes of proteins that are typically incompatible with other displays … We anticipate that dCas9-based display and PICASSO will be useful for the investigation of customized peptide and protein libraries for many additional applications, including multiplexed diagnostics, enzyme substrate discovery, and protein evolution and design experiments.”