Engineered Cascade Complexes for Eukaryotic Genome Editing

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Solution Overview

Problem

Type I CRISPR-Cas systems have limited use in eukaryotic genome engineering due to difficulties in heterologous expression of the Cascade complex and the way they cleave DNA targets.

Innovation Solution

Engineered Type I CRISPR-Cas effector complexes are developed, comprising specific protein components, modified guide polynucleotides, and fusion proteins like Cas8-FokI, to enhance genome editing capabilities in cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If Type I CRISPR-Cas systems are used for genome engineering, then DNA targeting capability is provided, but heterologous expression of the Cascade complex is difficult

Engineering Contradiction:

ImproveDNA targeting capabilityVSAvoidheterologous expression of Cascade complex

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The Cascade complex is divided into separate subunit proteins that can be individually expressed and then assembled with guide RNA in trans, bypassing the need for heterologous expression of the entire complex

Inventive Principle:

Principle #1Segmentation

2Reliability

If Type I CRISPR-Cas systems are used for genome engineering, then DNA cleavage activity is provided, but the cleavage mechanism is unsuitable for eukaryotic applications

Engineering Contradiction:

ImproveDNA cleavage activityVSAvoidsuitability for eukaryotic applications

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The Cas8 subunit protein is fused to the FokI nuclease domain, combining the DNA binding capability of the CRISPR system with the cleavage activity of FokI, which functions independently of the problematic Type I cleavage mechanism

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The FokI nuclease acts as an intermediary that provides DNA cleavage activity compatible with eukaryotic systems, replacing the native Type I cleavage mechanism while maintaining target specificity through the CRISPR guide RNA-Cas8 complex

Inventive Principle:

Principle #24Intermediary (Mediator)

3Productivity

If engineered Type I CRISPR-Cas effector complexes are developed, then genome editing efficiency is improved, but system complexity increases

Engineering Contradiction:

Improvegenome editing efficiencyVSAvoidsystem complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The engineered Cas8-FokI fusion protein serves multiple functions: it provides DNA binding through the CRISPR guide RNA complex, recruits the FokI nuclease to the target site, and enables programmable genome editing with improved efficiency across different target sequences

Inventive Principle:

Principle #6Universality (Multi-functionality)

Data Source

PatentUS12227776B2Engineered cascade components and cascade complexes

Publication Date: 2025.02.18 CARIBOU BIOSCIENCES INC

AI summary

The present disclosure provides engineered Class 1 Type I CRISPR-Cas (Cascade) systems that comprise multi-protein effector complexes, nucleoprotein complexes comprising Type I CRISPR-Cas subunit proteins and nucleic acid guides, polynucleotides encoding Type I CRISPR-Cas subunit proteins, and guide polynucleotides. Also, disclosed are methods for making and using the engineered Class 1 Type I CRISPR-Cas systems of the present invention.