Genome Editing Complex with Proteolysis Tag for Reduced Toxicity
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Solution Overview
Problem
Conventional genome editing vectors impose a heavy burden on bacterial hosts, leading to instability and high toxicity, resulting in non-specific mutations and cell death, particularly when using uracil DNA glycosylase inhibitors, which increases toxicity and non-specific mutation rates.
Innovation Solution
A complex comprising a nucleic acid sequence-recognizing module linked with a proteolysis tag, such as a peptide with hydrophobic amino acid residues, is used to reduce non-specific mutations by shortening the half-life of the genome editing complex, allowing for targeted DNA alteration without relying on host-dependent factors like RecA, thereby stabilizing the vector and reducing toxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional genome editing vectors are used to achieve targeted DNA alteration, then editing function is provided, but vector stability deteriorates and toxicity increases causing non-specific mutations and cell death
Solution Approach 1:
The patent applies dynamics by making the genome editing complex transient through proteolysis tag-mediated degradation. The complex is dynamically controlled to be present only temporarily during editing, then degraded by host proteases, preventing accumulation that causes toxicity and instability. This resolves the contradiction by allowing editing function when needed while eliminating the complex afterward to maintain vector stability.
Solution Approach 2:
The patent changes the temporal parameter of complex presence by introducing proteolysis tags that control protein half-life. This parameter change from persistent to transient expression allows the system to achieve editing function temporarily while avoiding the stability issues caused by prolonged presence of the complex, thus resolving the contradiction between editing efficacy and vector stability.
2Productivity
If uracil DNA glycosylase inhibitors are used to increase mutation efficiency, then editing efficiency improves, but toxicity increases causing cell death and non-specific mutations
Solution Approach 1:
The patent applies dynamics by controlling the temporal presence of the genome editing complex through proteolysis. The complex is present only transiently to perform editing, then degraded, which reduces cumulative toxicity while maintaining editing efficiency. This dynamic control resolves the contradiction between achieving high mutation efficiency and avoiding toxicity-induced cell death.
Solution Approach 2:
The patent treats the genome editing complex as a disposable, short-lived entity by introducing proteolysis tags. The complex performs its editing function and is then degraded by host proteases, eliminating the need for long-term persistence. This approach maintains editing efficiency while reducing toxicity, as the complex does not accumulate to harmful levels.
3Productivity
If genome editing complex is present in high amount to achieve high editing efficiency, then mutation efficiency improves, but non-specific mutations increase due to toxicity
Solution Approach 1:
The patent applies dynamics by controlling the temporal presence of the editing complex through proteolysis. The complex is present transiently at sufficient concentrations to achieve editing efficiency, then degraded to prevent off-target effects. This dynamic control resolves the contradiction between achieving high editing efficiency and maintaining mutation specificity.
Solution Approach 2:
The patent treats the editing complex as a short-lived, disposable entity that performs its function and is then degraded. This transient presence allows high local concentration for efficient editing without prolonged exposure that would cause non-specific mutations, thus resolving the contradiction between editing efficiency and mutation specificity.
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 approach enables stable genome editing with reduced non-specific mutations and high mutation efficiency at the target site, applicable to a wide range of bacteria, while minimizing off-target effects and cell death.
Implementation Method 1
a peptide showing a shortened half-life of a protein when added to a complex for genome editing
Data Source
AI summary
The present invention provides a complex containing a nucleic acid sequence-recognizing module and a proteolysis tag, wherein the module is linked to the proteolysis tag, the module specifically binds to a target nucleotide sequence in a double stranded DNA, and the tag consists of (i) a peptide containing 3 hydrophobic amino acid residues at the C-terminal, or (ii) a peptide containing 3 amino acid residues at the C-terminal wherein at least a part of the amino acid residues is substituted by serine