Sep 13, 2021

In a brand new application for CRISPR/Cas editing tools, a team at Harvard Medical School, Boston, MA, has developed a CRISPR/Cas9 ribonucleoprotein-based peptide display approach. With a new technique, they call peptide immobilization by Cas9-mediated self-organization “PICASSO,” this approach aims to facilitate the peptide library analysis workflow.

This innovative application of CRISPR/Cas tools relies on using a modified Cas9 that is both inactivated (dCas9) and fused to a peptide sequence. Guided by RNA barcodes (gRNA), the peptide fused-dCas9 is delivered onto a DNA microarray where the ribonucleoprotein complex self-assembles to complementary DNA sequences. Following the addition of samples of interest, such as antibodies from human serum, fluorescently labeled antibodies enable the readout. Combined with saturation mutagenesis libraries, this approach promises to support high-throughput antibody-epitope binding assays to facilitate antibody-drug and vaccine development.

Technology Behind DNA Microarrays

DNA microarray design flexibility is afforded by GenScript's semiconductor technology, which enables precise DNA synthesis and printing at predetermined positions on a solid surface. Because microarrays consist of single-stranded DNA oligos, Barber and colleagues implemented a primer annealing and extension step to generate double-stranded DNA microarrays, enabling fused-dCas9 complex specific binding.

DNA synthesis by semiconductor technology. Precise DNA synthesis is enabled by semiconductor technology, electrochemical techniques, and phosphoramidite oligo synthesis chemistry. Electrochemically generated acid is used at individual electrodes with standard phosphoramidite chemistries to synthesize unique DNA probes. 1. Protected nucleotides are bound to an adhesion layer above electrodes. 2. Nucleotides are deprotected when an electrode is activated. 3-5. A new phosporamidite is added, and the amidite couples only to deprotected nucleotides on the array. Repeating these steps generates oligonucleotide sequences.

Marcelo Caraballo, Senior Scientist at GenScript, shared a recent unique design used for testing their new GenScript's miniature semiconductor HD (8 million oligos) chip.

Picasso Technique Proof of Concept

In a playful template DNA microarray designed by leveraging the positioning of on-target and off-target DNA sequences, Barber and colleagues replicated the bull’s head from a Pablo Picasso’s sketch, artfully demonstrating the conserved binding specificity of fused-dCas9. They found that peptide fused-dCas9 retained DNA binding properties typical of dCas9 and that peptide sequences did not affect gRNA or DNA binding, supporting the feasibility of dCas9 based peptide display and the PICASSO approach.

To test the utility of this approach for the analysis of antibody-epitope interactions, the investigators focused on two human diseases, including influenza and COVID-19. The binding of convalescent serum derived-antibodies to saturation mutagenesis libraries of influenza A or SARS-CoV-2 peptides was evaluated in PICASSO microarrays. Significantly, antibody-epitope binding profiles revealed by PICASSO correlated nicely with previous phage display findings.

Advantages of CRISPR/Cas9 Based Peptide Display

This newly developed CRISPR/Cas-based Peptide Library Display platform presents several advantages. First, by encoding the fused-dCas9 and gRNA within a single vector, the team found that it is possible to multiplex the expression and purification of CRISPR/Cas complexes, allowing the use of a single E. coli culture. Second, the self-assembly of gRNA barcoded and pooled ribonucleotide complexes onto DNA microarrays significantly expedites and facilitates peptide library analysis. Lastly, Barber and colleagues point out that the option of reusing DNA microarrays following protease treatment makes PICASSO a more economical approach than synthetic peptide microarrays.

Reference

• Barber KW, Shrock E, Elledge SJ. CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays. Mol Cell. (2021). doi: 10.1016/j.molcel.2021.07.027.