Cas1 Cas2 Integration of Donor DNA into Eukaryotic Target Genomes

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Solution Overview

Problem

Current methods lack efficient means to integrate foreign DNA into target DNA molecules, particularly in eukaryotic cells, where CRISPR systems are not native, and existing CRISPR systems require specific loci and structures for functionality.

Innovation Solution

The use of Cas1 and Cas2 proteins, along with an integration host factor (IHF) protein, to facilitate the integration of a donor DNA molecule into a target DNA molecule, especially in eukaryotic cells, by contacting the target DNA with a linear donor DNA and Cas1 protein, potentially in the presence of Cas2 and IHF, to achieve specific integration without requiring native CRISPR loci or structures.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If CRISPR systems are used for DNA integration, then integration capability is provided, but native CRISPR loci and structures are required which limit applicability in eukaryotic cells

Engineering Contradiction:

Improveapplicability in eukaryotic cellsVSAvoidrequirement for native CRISPR loci and structures

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent extracts the essential integration function from the complete CRISPR system by isolating Cas1 and Cas2 proteins, removing the requirement for native CRISPR loci and repetitive sequence structures. This extraction allows the integration capability to function independently in eukaryotic cells without the complex prokaryotic CRISPR machinery

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The Cas1-Cas2 protein complex is demonstrated to perform DNA integration function universally across different cell types including eukaryotic cells, without requiring cell-type-specific CRISPR loci. The system achieves multi-functionality by integrating donor DNA at AT-rich regions in various genomic contexts

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Productivity

If existing CRISPR systems are employed, then foreign DNA detection and destruction is achieved, but integration of foreign DNA into target DNA is not facilitated

Engineering Contradiction:

ImproveDNA integration efficiencyVSAvoidfunctional capability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The Cas1-Cas2 protein complex serves as an intermediary mechanism that bridges the gap between foreign DNA detection and actual integration. These proteins mediate the transfer and insertion of donor DNA sequences into target genomic DNA at specific AT-rich regions, enabling productive integration that existing CRISPR systems cannot achieve

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS11053271B2Methods and compositions for nucleic acid integration

Publication Date: 2021.07.06 RGT UNIV OF CALIFORNIA

AI summary

The disclosure provides methods and compositions for the integration (insertion) of a donor DNA molecule into a target DNA molecule. In general, the methods include contacting a target DNA molecule with a linear donor DNA molecule and a Cas 1 protein, where the target DNA molecule includes an AT-rich region (e.g., in some cases positioned 5 and within 50 nucleotides of a region that forms a DNA cruciform structure), where the contacting is not in a bacterial or archaeal cell (e.g., the contacting is in vitro outside of a cell, inside of a eukaryotic cell, etc.), and provides for integration of the donor DNA molecule into the target DNA molecule.