Repeatable Directed Endonucleases for Offset DNA Cleavage
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Solution Overview
Problem
Current directed endonucleases are limited in their ability to make specific, repeatable cleavages at targeted DNA sequences, which hinders precise genome engineering and data recording applications.
Innovation Solution
Development of repeatable directed endonucleases (RDEs) that cleave DNA at a fixed offset from their recognition sequence, allowing for repeated localization and further cleavage while preserving the recognition sequence, enabling efficient genome engineering and programming of cellular events.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If directed endonucleases cleave DNA at their recognition sequence, then specific DNA cleavage is achieved, but the recognition sequence is destroyed and cannot be reused for further cleavage
Solution Approach 1:
The invention separates the DNA recognition function from the DNA cleavage function. The directed endonuclease is engineered to recognize and bind to a specific DNA sequence without cleaving at that exact location, instead cleaving at an offset position. This segmentation allows the recognition sequence to remain intact and reusable while still achieving precise cleavage at the desired location through the offset mechanism.
2Manufacturing precision
If directed endonucleases are used for genome engineering, then specific DNA modifications are achieved, but the ability to perform repeated modifications at the same locus is limited
Solution Approach 1:
The invention performs preliminary action by designing the endonuclease to preserve the recognition sequence during the first cleavage event. By cleaving at an offset position rather than within the recognition sequence itself, the recognition site remains intact and available for subsequent binding events. This preliminary preservation of the recognition sequence enables multiple rounds of cleavage and modification at the same genomic locus, extending the duration and repeatability of the genome engineering action.
3Manufacturing precision
If the recognition sequence is cleaved, then DNA modification is achieved, but the recognition sequence is lost preventing re-localization
Solution Approach 1:
The invention introduces an intermediary mechanism through the offset cleavage design. The directed endonuclease acts as an intermediary that binds to the recognition sequence but performs its cleavage function at a displaced location. This intermediary approach allows the recognition sequence to serve as a stable docking site that is not consumed by the cleavage reaction, enabling the endonuclease to re-localize repeatedly to the same recognition sequence for additional cleavage events if needed.
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
RDEs facilitate precise and repeatable DNA modifications, enhancing genome engineering capabilities and allowing for controlled biochemical pathways and delayed cell death, thereby improving biomanufacturing efficiency and genetic manipulation.
Implementation Method 1
a DNA-recognition domain (DRD) of any directed endonuclease or directed endonuclease binding domain
Implementation Method 2
a nuclease domain linked to the DRD via a linking domain wherein the nuclease domain is capable of cleaving the target nucleic acid
Data Source
AI summary
The invention provides compositions and methods for repeatable directed endonucleases (RDEs) and methods for repeatedly, and specifically cleaving DNA offset from the RDE's DNA recognition sequence on the target nucleic acid rather than within the DNA recognition sequence. Conservation of the recognition sequence of the target nucleic acid enables for re-localization of an RDE back to the DNA recognition sequence for further cleavage. The RDEs and methods of the invention are useful in applications including, but not limited to, recording data into a genome, timing the order of biochemical pathway events, efficient genome engineering and encoding lagged cellular death.