Reprogrammable IsrB Nucleases for Scalable Genome Targeting
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Solution Overview
Problem
Current genome-editing technologies lack robust, affordable, and scalable strategies for targeted genome perturbations that can efficiently target multiple positions within the genome.
Innovation Solution
Development of a non-naturally occurring composition comprising an IsrB polypeptide with a split Ruv-C nuclease domain and an ωRNA molecule with a reprogrammable spacer sequence, capable of forming a complex to direct the polypeptide to a target polynucleotide, along with optional functional domains for various activities such as nuclease, transposase, or recombinase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-Cas systems are used for genome editing, then targeted genome perturbations can be achieved, but the systems are complex and not easily scalable to multiple positions
Solution Approach 1:
The patent segments the genome editing function into separate components: a simplified nuclease domain (RuvC or HNH) that performs the cutting function, and separate guide RNA molecules (ωRNA and δRNA) that provide targeting. This segmentation allows each component to be optimized independently and facilitates scaling to multiple target positions by simply changing the guide sequences rather than redesigning the entire system.
Solution Approach 2:
The patent creates a universal platform where the same nuclease domain can be used with different guide RNA combinations to target multiple positions in the genome. The standardized architecture with conserved structural elements and variable guide sequences allows the system to function universally across different target sites, enabling scalable genome editing applications.
2Adaptability or versatility
If existing genome-editing techniques are used, then some targeted modifications are possible, but they lack robustness and affordability for multiple position targeting
Solution Approach 1:
The patent extracts the essential genome editing function from the complex CRISPR-Cas system, retaining only the critical nuclease domain and guide RNA components. By removing unnecessary elements and focusing on the core mechanism, the system achieves both robustness through simplified design and versatility through the ability to easily reprogram guide sequences for different target positions.
Solution Approach 2:
The patent enables versatile multi-position targeting by allowing changes in the guide RNA sequence parameters while keeping the nuclease domain structure constant. This parameter change approach—modifying only the guide sequences—provides a simple, affordable method to adapt the system to different target positions while maintaining reliable editing function through the conserved nuclease architecture.
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
Enables efficient and targeted modification of polynucleotides, including cleavage and insertion of donor sequences, facilitating precise genetic edits and corrections, with versatility in targeting multiple genomic locations.
Implementation Method 1
an ωRNA molecule comprising a scaffold and a reprogrammable spacer sequence, the ωRNA molecule capable of forming a complex with the IsrB polypeptide and directing the IsrB polypeptide to a target polynucleotide
Implementation Method 2
an IsrB polypeptide comprising a split Ruv-C nuclease domain comprising RuvC-I, RuvC-II, and RuvC-III subdomains
Data Source
AI summary
Systems, methods and compositions for targeting polynucleotides are detailed herein. In particular, engineered DNA-targeting systems comprising IsrB polypeptides, novel IsrB nucleases and reprogrammable targeting nucleic acid components and methods and application of use are provided.