TteAgo Protein DNA Cleavage for Gene Editing
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Solution Overview
Problem
Current studies have focused on the RNA interference pathway of eukaryotic Argonaute proteins, but their interaction with DNA and potential for gene editing in mammalian cells remains underexplored, particularly due to the lack of documented DNA cleavage activity.
Innovation Solution
The disclosure provides an Argonaute protein from Thermomycetes thermophilus, TteAgo, with demonstrated DNA cleavage activity, which can form a complex with guide molecules to specifically cleave target nucleic acids, including RNA and DNA, and is applicable for gene editing in mammalian cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If eAgo protein from higher animals and plants is used, then RNAi pathway activity is maintained, but DNA cleavage capability and gene editing effectiveness are lost
Solution Approach 1:
The patent segments the Argonaute protein family by identifying distinct functional subtypes: eAgo for RNAi and TteAgo for DNA cleavage. By separating these functions into different protein sources (eukaryotes vs. thermophilic eukaryotes), the patent enables selective application of each protein type for its optimal function, resolving the contradiction between maintaining RNAi activity and achieving DNA cleavage capability
Solution Approach 2:
TteAgo protein exhibits multi-functionality by demonstrating both DNA cleavage activity and potential RNAi activity. The patent shows that TteAgo can function as a guide RNA/DNA-dependent endonuclease while also potentially participating in RNA interference pathways, making it a universal tool that can address both RNA and DNA targeting needs in gene editing applications
2Productivity
If eAgo protein with high RNase activity is used, then RNA targeting efficiency is improved, but DNA interaction and cleavage activity are insufficient
Solution Approach 1:
The patent introduces TteAgo as an intermediary protein that mediates DNA cleavage through guide RNA or guide DNA molecules. Unlike eAgo proteins that primarily interact with RNA targets, TteAgo serves as a specialized intermediary that facilitates DNA recognition and cleavage while maintaining guide molecule dependency, thus enabling reliable DNA cleavage activity without compromising RNA targeting efficiency in applications where both are needed
3Adaptability or versatility
If eAgo protein is used for gene editing, then RNA interference function is achieved, but effective gene editing in mammalian cells cannot be realized
Solution Approach 1:
The patent applies parameter changes by selecting TteAgo from Thermomycetes thermophilus, a thermophilic eukaryote, which possesses optimal biochemical parameters for mammalian cell applications. The protein's thermostability and nuclease activity parameters make it more suitable for gene editing in mammalian cells compared to mesophilic eAgo proteins, thereby improving gene editing effectiveness while retaining RNA interference function
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
The TteAgo protein enables site-specific modification of genetic materials, offering a new tool for gene editing and modification, with high specificity and efficiency, and the potential to apply in various biotechnological fields.
Implementation Method 1
combine with a single-stranded RNA molecule as a small guide specifically recognizing a complementary RNA target
Implementation Method 2
directly cleave the target through the inherent nuclease activity of some eAgo enzymes
Data Source
AI summary
An Argonaute protein from eukaryotes and an application thereof are provided. An amino acid sequence of the Argonaute protein is shown in SEQ ID NO: 1 or has at least 50% sequence identity with the sequence shown in SEQ ID NO: 1. The specific cleavage activity of the eukaryotic Argonaute protein on DNA is first proved, and an experimental proof for the study of interaction between the eukaryotic Argonaute protein and DNA is provided. In addition, polypeptides, nucleic acids, expression vectors, compositions, kits, and methods used therein can carry out site-specific operation on intracellular and extracellular genetic materials and can be effectively applied in many fields of biotechnology, providing a new tool for gene editing, modification, and molecular detection of Argonaute polypeptides based on eukaryotic sources.