CRISPR Protein Barcoding for Microarray Assembly
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current protein microarrays and display platforms are labor-intensive and costly to construct, requiring individual purification of thousands of proteins for spotting, and there is a need for efficient and customizable protein libraries for disease diagnostics and therapeutic discovery.
Innovation Solution
The use of Cas-containing fusion proteins, including catalytically inactive Cas9, Cas12a, Cas13, or Cas14 proteins linked with a single guide RNA (sgRNA) and a protein of interest, which self-assemble on a microarray or non-microarray surface through specific hybridization with DNA probes, allowing for efficient immobilization and customization of protein libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional protein microarrays are constructed by individual purification and spotting of thousands of proteins, then protein library capacity and complexity can be achieved, but the construction process becomes labor-intensive and costly
Solution Approach 1:
The patent uses DNA barcodes as information copies to represent proteins in a library. Instead of physically manipulating thousands of purified proteins, the system creates DNA barcode sequences that encode protein identity information. These barcodes can be synthesized and manipulated digitally, dramatically reducing labor while maintaining full library capacity. The DNA barcode serves as an information duplicate that replaces the need for physical protein handling.
Solution Approach 2:
The patent replaces the mechanical process of protein purification and spotting with a biochemical system based on CRISPR-Cas9 guided by DNA barcodes. The mechanical manipulation of physical proteins is substituted with molecular recognition events where Cas9 proteins guided by sgRNAs (containing barcode information) automatically localize to correct positions on the microarray through sequence-specific DNA binding, eliminating manual spotting operations.
2Loss of time
If CRISPR-based self-assembly is used to construct protein microarrays, then construction time and cost are reduced, but precise positioning and localization accuracy must be maintained
Solution Approach 1:
The patent introduces DNA barcodes as intermediary molecules that mediate between the protein library and the microarray positions. The DNA barcode sequences serve as address codes that the CRISPR system reads to determine localization positions. This intermediary layer enables automatic, precise positioning through sequence complementarity between barcodes and target sites, replacing manual positioning while maintaining accuracy.
Solution Approach 2:
The patent performs preliminary encoding of protein identity information into DNA barcode sequences before the microarray construction process. The barcode sequences are designed in advance to complement specific target DNA sequences at predetermined microarray positions. This preliminary information encoding allows the self-assembly process to automatically achieve correct positioning without real-time intervention, reducing construction time while preserving precision.
3Quantity of substance
If in vitro protein display techniques are used for large-scale protein studies, then protein library capacity is increased, but the complexity of library design and customization increases
Solution Approach 1:
The patent creates a universal DNA barcode system that can represent any protein in the library using a standardized encoding scheme. The same CRISPR-Cas9 machinery and barcode reading protocol work for all proteins in the library, regardless of their specific functions or structures. This universal interface simplifies library design because all proteins are handled through the same DNA barcode mechanism, reducing complexity despite increased library capacity.
Solution Approach 2:
The patent changes the fundamental parameter of protein identification from physical protein characteristics to digital DNA barcode sequences. By encoding protein identity as nucleotide sequences rather than relying on physical protein properties, the system enables computational design and manipulation of library members. This parameter transformation simplifies library customization because DNA sequences can be easily designed, synthesized, and modified using standard molecular biology techniques.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables rapid and cost-effective generation of complex protein microarrays, facilitating multiplexed protein studies and diagnostics by allowing proteins to self-assemble on user-defined positions, reducing off-target localization and enhancing the efficiency of protein library construction and analysis.
Implementation Method 1
The sgRNA includes a unique nucleotide sequence complementary to a target sequence of a DNA probe
Data Source
AI summary
Biotechnological innovations have vastly improved the capacity to perform large-scale protein studies. The production and interrogation of custom protein libraries has proven important for a plethora of biological applications including multiplexed disease diagnostics, therapeutic antibody discovery, and directed evolution. The present invention relates to methods and compositions for use in making Cas-related fusion protein libraries barcoded with sgRNA sequences for applications in protein studies and for protein self-assembly on surfaces.