CRISPR Base Editors for Unnatural Amino Acid Incorporation
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Solution Overview
Problem
Current methods lack programmable tools for unbiased high-throughput interrogation of the proteome in living cells, limiting the study of cellular systems and disease-relevant biological processes, as existing tools are primarily designed for individual proteins or small collections and are not applicable to the entire proteome.
Innovation Solution
The integration of unnatural amino acids into proteins using CRISPR/Cas9-based base-editing technology, specifically through stop-codon or rare codon suppression, enables the incorporation of these amino acids into virtually any protein by transforming target codons into stop or rare codons using guide RNA or ssDNA complexed with Cas9 nucleobase editors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR/Cas9-based base-editing technology is used to incorporate unnatural amino acids into proteins, then proteome-wide interrogation capability is enabled, but device complexity increases due to the need for guide RNA/ssDNA and Cas9 nucleobase editor components
Solution Approach 1:
The CRISPR/Cas9 base-editing system is designed to be universally applicable across the entire proteome by using programmable guide RNA or ssDNA sequences that can target any desired codon position in any protein-coding gene. The same Cas9 nucleobase editor component can be reused with different guides to achieve proteome-wide interrogation, making the system multi-functional and highly adaptable rather than requiring protein-specific tools for each application
Solution Approach 2:
The guide RNA or ssDNA acts as an intermediary component that bridges the user's research intent and the Cas9 nucleobase editor's editing function. By designing different guide sequences, researchers can programmably direct the editing machinery to specific target codons without modifying the Cas9 editor itself, thus managing system complexity through a modular intermediary layer
2Productivity
If stop-codon or rare codon suppression is used to incorporate unnatural amino acids, then high-throughput proteome interrogation is enabled, but manufacturing precision requirements increase due to the need for precise codon transformation
Solution Approach 1:
The patent replaces traditional mechanical or chemical methods of protein modification with a programmable biochemical system. The Cas9 nucleobase editor uses guide RNA-programmed DNA binding to precisely locate target codons, and the deaminase domain catalyzes specific C-to-U base conversions to transform codons (e.g., CAG→UAG for amber stop, or rare codons like AGG→UGG). This biochemical precision system enables high-throughput applications because once the guide RNA is designed, the transformation is automatically precise without manual intervention for each target
Solution Approach 2:
The system changes the biochemical parameter of codon sequence by catalyzing deamination reactions that convert specific bases. By controlling which cytosine bases are deaminated (through guide RNA programming), the system precisely transforms codons to incorporate unnatural amino acids at desired positions. This parameter change approach allows high-throughput screening because multiple codon transformations can be achieved by simply changing the guide RNA sequence parameters rather than redesigning the entire editing mechanism
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 allows for proteome-wide interrogation of protein functions, introducing new functional groups and enabling high-throughput research and therapeutic target discovery by incorporating unnatural amino acids into any protein in a programmable manner.
Implementation Method 1
changing a target codon to a stop codon or a rare codon via deamination of a cytosine (C) base
Data Source
AI summary
Provided herein are systems, compositions, and methods for the incorporation of unnatural amino acids into proteins via nonsense suppression or rare codon suppression. Nonsense codons and rare codons may be introduced into the coding sequence of a protein of interest using a CRISPR/Cas9-based nucleobase editor described herein. The nucleobase editors are able to be programmed by guide nucleotide sequences to edit the target codons in the coding sequence of the protein of interest. Also provided are application enabled by the technology described herein.