Inducible Genome Editing Construct for Non-Leaky Gene Control
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genome editing systems require multiple constructs for targeted modification, leading to leaky gene expression and inefficiencies, lacking precise and spatial-temporal control.
Innovation Solution
A single-engineered nucleic acid construct with a promoter, regulatory protein, inducible promoter, insulators, and DNA-binding domain recognition sequences, enabling spatially and temporally controlled genome editing through non-homologous end-joining, without introducing vector material.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple independent constructs are used for targeted genome modification, then the genome editing function can be achieved, but the gene expression becomes leaky and the system efficiency decreases
Solution Approach 1:
The patent combines multiple independent constructs into a single integrated construct that contains both the regulatory protein expression cassette and the target gene expression cassette. This single construct is introduced into the genome as one unit, eliminating the need for multiple separate constructs while maintaining the genome editing function and reducing leaky expression.
2Reliability
If multiple independent constructs are used for targeted modification, then the genome editing capability is achieved, but precise spatial-temporal control is lost
Solution Approach 1:
The patent merges the regulatory protein expression cassette and target gene expression cassette into a single construct with an inducible promoter system. This allows both genes to be controlled by the same inducible mechanism (e.g., tetracycline, doxycycline), enabling precise spatial-temporal control where gene expression is activated only when and where the inducer is present.
Solution Approach 2:
The patent implements a dynamic control system using inducible promoters that can be turned on or off in response to external signals (inducers). This allows the system to adapt gene expression levels dynamically based on experimental conditions, providing precise temporal and spatial control over genome editing activity.
3Productivity
If a single construct is used with inducible promoter, then tight regulation and non-leaky expression are achieved, but the construct design becomes more complex
Solution Approach 1:
The patent segments the single construct into distinct functional cassettes: a regulatory protein expression cassette, a target gene expression cassette, and insulator elements. Each cassette is designed with specific boundaries and functions, making the overall complex construct manageable through modular organization while maintaining tight regulation and non-leaky expression.
Solution Approach 2:
The patent introduces insulator elements as intermediary sequences between the regulatory and target gene cassettes. These insulators act as boundary elements that prevent unwanted interactions and ensure independent regulation of each cassette, simplifying the design by creating clear functional boundaries within the single construct.
4Ease of manufacture
If vector material is introduced with the construct, then the construct can be delivered, but unwanted vector DNA remains in the genome
Solution Approach 1:
The patent extracts and removes vector material from the final genomic integration. The construct is designed to be delivered in a form that excludes plasmid or viral vector sequences, ensuring that only the desired construct DNA is incorporated into the genome without unwanted vector backbone or regulatory elements remaining.
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
Provides tightly regulated and efficient genome editing with precise gene expression, minimizing leakiness and enhancing integration efficiency.
Implementation Method 1
site-specific integration into a genome via the non-homologous end-joining (NHEJ) pathway
Implementation Method 2
an inducible promoter operably linked to a nucleic acid encoding an enzyme that cleaves nucleic acid... wherein activity of the inducible promoter is modulated by the regulatory protein
Implementation Method 3
at least two insulators (e.g., mammalian insulators) located downstream from (a) and upstream from (b); at least one insulator located downstream from (b) and upstream from (a)
Data Source
AI summary
The present disclosure is directed, in some embodiments, to compositions and methods for inducible modification of a cell genome.