CRISPR-Cas9 Genome Editing in Archaea

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Solution Overview

Problem

Current genetic tools are inefficient and time-consuming for manipulating Archaea, particularly methanogenic organisms like Methanosarcina, due to slow growth rates and fastidious cultivation requirements, limiting the pace of genetic studies.

Innovation Solution

Development of RNA-guided DNA endonuclease-mediated genome editing tools using CRISPR-Cas systems, specifically utilizing the native homology-directed repair (HDR) machinery in Archaea for precise insertion and deletion of genes, and co-expression of non-homologous end-joining (NHEJ) machinery to enable efficient and rapid genetic manipulation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If traditional genome manipulation techniques are used in Archaea, then genetic studies can be conducted, but the process is time-consuming and inefficient due to slow growth rates and fastidious cultivation requirements

Engineering Contradiction:

Improvepace of genetic studiesVSAvoidtime needed to construct mutants

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The patent introduces CRISPR-Cas systems as intermediary tools that mediate genome editing in Archaea. The RNA-guided DNA endonuclease (Cas9) acts as a mediator that recognizes target sequences via sgRNA and introduces precise cuts, enabling efficient genome manipulation without relying on traditional slow methods. The system includes intermediary components like homology-directed repair templates and NHEJ machinery that facilitate precise genetic modifications.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent replaces traditional mechanical/genetic manipulation methods (such as conjugation, transformation with selectable markers) with RNA-guided molecular recognition and cutting systems. The CRISPR-Cas system uses RNA-DNA hybridization and enzymatic cleavage to achieve precise genome editing, substituting cumbersome traditional methods with more efficient molecular biology techniques.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Manufacturing precision

If CRISPR-Cas systems are introduced for genome editing, then precision and efficiency of genetic manipulation improve, but the system complexity increases

Engineering Contradiction:

Improveprecision of gene insertion and deletionVSAvoidcomplexity of genome editing system

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent employs a universal CRISPR-Cas system that can target multiple genomic locations using different sgRNAs. The same Cas9 protein and basic system architecture can be used for various genome editing applications (insertions, deletions, substitutions) by simply changing the guide RNA sequences, providing multi-functionality without requiring separate systems for each application.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent segments the genome editing function into modular components: the Cas9 endonuclease, the sgRNA guide sequences, the homology-directed repair templates, and the NHEJ machinery. This segmentation allows each component to be optimized independently and facilitates precise control over the editing process, improving precision while managing system complexity through modularity.

Inventive Principle:

Principle #1Segmentation

3Productivity

If multiple mutations are introduced simultaneously, then productivity of genetic studies increases, but the risk of off-target activity increases

Engineering Contradiction:

Improvesimultaneous introduction of multiple mutationsVSAvoidoff-target activity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent implements feedback control through careful design of sgRNA specificity and validation of editing outcomes. The system includes mechanisms to verify on-target editing and assess off-target effects, allowing researchers to adjust guide RNA sequences and experimental parameters to minimize harmful off-target activity while maintaining the ability to introduce multiple mutations simultaneously.

Inventive Principle:

Principle #23Feedback

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

This approach allows for rapid and precise genetic manipulation, reducing the time needed to construct mutants by half, enabling simultaneous introduction of multiple mutations and precise gene modifications without off-target activity, thus facilitating advanced genetic studies in Archaea.

Implementation Method 1

A DNA molecule can comprise a single-guide RNA (sgRNA) sequence that is complementary to a target sequence in a polynucleotide

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 2

an RNA-guided DNA endonuclease that binds to the target sequence in the presence of a protospacer adjacent motif (PAM), on the non-targeted DNA strand, thereby causing a double-strand break

Methodology Applied

Scientific EffectEnzymatic cleavage: Enzyme

Implementation Method 3

utilizing the native homology-directed repair (HDR) machinery in Archaea for precise insertion and deletion of genes

Methodology Applied

Scientific EffectHomologous recombination:

Implementation Method 4

co-expression of non-homologous end-joining (NHEJ) machinery to enable efficient and rapid genetic manipulation

Methodology Applied

Scientific EffectNon-homologous end joining:

Data Source

PatentUS20240043876A1Genome editing in archaea

Publication Date: 2024.02.08 THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS

AI summary

Methods of RNA-guided DNA endonuclease-mediated genome editing in Bacteria and Archaea are provided.