Codon-Adapted Argonaute Protein for Precise Eukaryotic Genome Editing
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Solution Overview
Problem
Current genome editing tools, such as CRISPR/Cas systems, are error-prone and sequence-biased, and fail to achieve precise, non-bias, and error-free introduction of desired base changes, deletions, or insertions in eukaryotic cells, with limited efficiency in homologous recombination.
Innovation Solution
Development of novel codon-adapted Argonaute protein variants (ANAGO) derived from microbial Argonaute proteins, reengineered to use human-preferred codons, enabling precise genome editing in eukaryotic cells through homologous recombination directed genome editing (AISE) with minimal off-target events.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas systems are used for genome editing in eukaryotic cells, then genome editing capability is achieved, but precision and error-free editing are compromised due to sequence bias and off-target events
Solution Approach 1:
The patent introduces ANAGO (codon-adapted Argonaute protein) as an intermediary tool that mediates genome editing through a different molecular mechanism than CRISPR/Cas. ANAGO uses guide DNA oligos to direct endonuclease activity to target sites, enabling precise editing without the sequence bias and off-target effects characteristic of CRISPR/Cas systems. This intermediary approach resolves the contradiction by providing an alternative pathway that achieves editing capability while maintaining high precision.
Solution Approach 2:
The patent modifies the codon usage parameters of the original microbial Argonaute protein to adapt it for eukaryotic expression. By changing the codon composition to match eukaryotic preferences, the protein maintains high fidelity and precision in eukaryotic cells while avoiding the harmful off-target effects. This parameter change enables reliable, precision editing in the target system.
2Productivity
If CRISPR/Cas systems are used for genome editing, then editing efficiency is improved, but manufacturing precision and error-free base changes are compromised
Solution Approach 1:
ANAGO serves as an intermediary that decouples editing efficiency from precision requirements. The system uses guide DNA oligos to direct ANAGO to specific target sites with high efficiency, while the Argonaute endonuclease domain ensures precise cleavage only at the intended location. This intermediary mechanism achieves both high productivity and high manufacturing precision simultaneously.
Solution Approach 2:
The ANAGO system segments the editing function into distinct components: guide DNA oligos for target recognition and direction, and the Argonaute endonuclease for precise cleavage. This segmentation allows each component to optimize its function independently, achieving both high efficiency in target finding and high precision in base change execution.
3Reliability
If microbial Argonaute proteins are used directly in eukaryotic cells, then endonuclease activity is retained, but expression efficiency is reduced due to codon usage differences
Solution Approach 1:
The patent changes the codon usage parameters of the microbial Argonaute protein coding sequence to match eukaryotic codon preferences. This parameter change dramatically improves translation efficiency and protein expression levels in eukaryotic cells while preserving the critical endonuclease active site residues and overall protein structure. The result is high productivity with maintained reliability of endonuclease function.
Solution Approach 2:
The patent applies local quality by selectively modifying only the codon composition of the coding sequence while leaving the amino acid sequence and critical functional domains unchanged. This localized modification approach improves expression efficiency without compromising the endonuclease activity that depends on specific amino acid residues and structural features.
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
ANAGO technology allows for precise genome editing in eukaryotic cells with high efficiency and no significant off-target events, enabling applications in gene therapy for treating diseases like chronic myelogenous leukemia and lowering LDL levels.
Implementation Method 1
mediating homologous recombination directed genome editing in eukaryotic cells
Implementation Method 2
capable of editing a target nucleic acid sequence within a eukaryotic cell
Data Source
AI summary
Disclosed herein are synthetic nucleic acids comprising a nucleic acid sequence that encodes a codon-Adapted Nuclear Argonaute protein (ANAGO) that is a species-specific to a eukaryote, and compositions comprising ANAGO and donor molecules for use in homologous recombination directed targeted gene editing in the eukaryote.