Modular Site-Directed Nuclease Assembly via Golden Gate Cloning

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Solution Overview

Problem

Current methods for developing novel combinations of site-directed nucleases and nucleotide-modifying enzymes lack standardized approaches for combinatorial composition, detection, and functional characterization, limiting the flexibility and customization of nucleic acid-modifying enzymes.

Innovation Solution

A method involving a sequence assembly system, such as Golden Gate cloning, where site-directed nucleases and nucleotide-modifying enzymes are provided as modular components with standardized cloning sites, allowing for the construction of multiple assemblies that can be expressed as single polypeptides, enabling the extension of functionality by joining additional modules.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If standardized sequence assembly systems are implemented for combinatorial composition of site-directed nucleases and nucleotide-modifying enzymes, then the flexibility and adaptability of nucleic acid-modifying enzymes are improved, but the device complexity and manufacturing precision requirements increase

Engineering Contradiction:

Improveflexibility and customization of nucleic acid-modifying enzymesVSAvoidcomplexity of sequence assembly system

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent divides the nucleic acid-modifying enzyme into separate functional modules: a site-directed nuclease module and a nucleotide-modifying enzyme module. These modules can be independently designed, synthesized, and assembled through standardized sequence assembly systems, enabling flexible recombination without increasing overall system complexity.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent creates a universal standardized sequence assembly system with common cloning sites and regulatory elements that can accommodate multiple different site-directed nucleases and nucleotide-modifying enzymes. This universal platform enables combinatorial assembly of various enzyme combinations using the same standardized procedures.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Productivity

If multiple combinatorial assemblies are constructed and characterized, then the functionality and productivity of nucleic acid modifiers are enhanced, but the time and resources required for detection and measurement increase

Engineering Contradiction:

Improveoutput of novel nucleic acid-modifying enzymesVSAvoidtime for detection and functional characterization

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The patent incorporates standardized cloning sites and regulatory elements into the module designs beforehand, allowing for rapid assembly and characterization. The preliminary standardization of these components enables high-throughput combinatorial assembly without proportionally increasing detection and measurement time.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent utilizes standardized regulatory elements with defined expression parameters that can be consistently applied across multiple assemblies. This standardization allows for predictable expression levels and simplifies the functional characterization process by reducing the need for parameter optimization for each new assembly.

Inventive Principle:

Principle #35Parameter changes

3Ease of manufacture

If modular components with standardized cloning sites are used, then the ease of manufacture and operation are improved, but the manufacturing precision and assembly accuracy requirements increase

Engineering Contradiction:

Improveease of construction of nucleic acid assembliesVSAvoidprecision of module assembly

Core Design Contradiction:

Ease of manufactureVSManufacturing precision

Solution Approach 1:

The patent uses standardized cloning sites as intermediary elements that mediate the assembly between different modular components. These cloning sites serve as universal connection points with defined sequences that facilitate precise and reliable joining of modules through standardized molecular biology techniques.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent employs homogeneous standardized regulatory elements and cloning sites across all modules, ensuring consistent assembly behavior and reducing variability in manufacturing precision requirements. This homogeneity allows for predictable and reproducible assembly outcomes across different combinatorial configurations.

Inventive Principle:

Principle #33Homogeneity

Data Source

PatentUS20230080513A1Compositions and methods for combinatorial assembly and screening for functional site-directed nucleotide modifiers

Publication Date: 2023.03.16 MERISTEM SPA

AI summary

The present invention discloses a method where a site-directed nuclease (e.g. Cas nuclease) as well as a nucleotide modifying enzyme (e.g. deaminase) are provided as modules of a sequence assembly system, such as a golden gate cloning system, comprising cloning sites and/or regulatory sites. The assembly system provides capacities for generating a polypeptide consisting of at least two standardised modules, each which can be composed of multiple peptide motifs, which optionally can be joined along with other standardised modules for extending the polypeptide's functionality further. New assemblies intended for site-directed nucleotide modification are also provided in this invention.