Engineered Cascade Complexes With Cas8-FokI Fusion for Eukaryotic 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 and DNA target cleavage mechanisms.
Innovation Solution
Engineering Type I CRISPR-Cas effector complexes with fusion proteins of Cas8 and FokI linked by linker polypeptides, along with modified guide polynucleotides, to enhance genome editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Type I CRISPR-Cas systems are used for genome engineering in eukaryotic cells, then DNA targeting capability is achieved, but heterologous expression difficulties and complex multi-protein assembly reduce ease of manufacture and reliability
Solution Approach 1:
The patent segments the Type I CRISPR-Cas system into separate expression units by fousing Cas8 with FokI nuclease. This segmentation allows the complex multi-protein Cascade complex to be expressed more easily in heterologous eukaryotic systems while maintaining the DNA targeting capability through the guide RNA-Cascade complex interaction and FokI-mediated cleavage.
Solution Approach 2:
The patent merges Cas8 subunit with FokI nuclease into a fusion protein. This merging simplifies the system by combining the scaffold function of Cas8 with the cleavage activity of FokI, eliminating the need for separate expression and coordination of multiple proteins while maintaining reliable genome editing in eukaryotic cells.
2Reliability
If native Type I CRISPR-Cas DNA cleavage mechanism is used, then DNA targeting is achieved, but the complex multi-step process reduces productivity
Solution Approach 1:
The patent combines the DNA binding function of the Cascade complex with the cleavage function of FokI in a unified system. The Cas8-FokI fusion protein allows the Cascade complex to guide the fusion protein to the target DNA sequence, where FokI directly cleaves the DNA, simplifying the multi-step native process into a more efficient single-step cleavage mechanism while maintaining targeting specificity.
3Reliability
If multiple Cas subunit proteins are expressed separately, then proper complex assembly is achieved, but device complexity increases
Solution Approach 1:
The patent segments the expression strategy by creating a focal point for complex assembly around the Cas8-FokI fusion protein. The Cascade complex still assembles with multiple subunits (Cas5, Cas6, Cas7, etc.) guided by the crRNA, but the critical cleavage function is consolidated in the fusion protein, reducing the number of separate expression components needed while maintaining proper assembly accuracy.
Solution Approach 2:
The Cas8 subunit in the fusion protein serves multiple functions: it is part of the Cascade complex structure, binds the crRNA guide, and provides the platform for FokI nuclease activity. This multi-functionality reduces the need for separate proteins and simplifies the overall system architecture while maintaining reliable complex assembly.
Data Source
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.