Modified Cascade Ribonucleoproteins for RNA-Guided Site-Specific Editing
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Solution Overview
Problem
There is a need for improved agents for sequence/site specific nucleic acid detection and/or cleavage in genetic engineering and genomic research, as existing methods like zinc finger nucleases require extensive protein engineering for each new DNA locus and lack specificity.
Innovation Solution
Development of a CRISPR-associated Cascade complex comprising Cas7, Cas5, and Cas6 protein subunits with additional amino acid sequences for nucleic acid or chromatin modifying, visualising, transcription activating, or transcription repressing activities, combined with an RNA molecule for targeted nucleic acid recognition and cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If zinc finger nucleases are used for sequence specific nucleic acid detection and/or cleavage, then site-specific modification can be achieved, but extensive protein engineering is required for each new DNA locus and specificity is insufficient
Solution Approach 1:
The Cascade complex is divided into multiple protein subunits (Cas7, Cas5, Cas6) that can be individually expressed and assembled. Each subunit has a specific function: Cas7 provides structural framework and nuclease activity, Cas5 binds crRNA, and Cas6 processes pre-crRNA. This segmentation allows modular optimization and simplifies the engineering process compared to creating entirely new zinc finger proteins for each target locus.
Solution Approach 2:
The Cascade complex uses a universal mechanism for target recognition through crRNA-guided binding. The same Cascade protein complex can target different DNA sequences by simply changing the crRNA guide sequence, eliminating the need for extensive protein re-engineering required by zinc finger nucleases for each new target locus.
2Reliability
If existing nucleic acid detection and cleavage methods are used, then basic functionality is achieved, but fidelity and accessibility to difficult loci are insufficient
Solution Approach 1:
The crRNA serves as an intermediary molecule that mediates between the Cascade protein complex and the target DNA sequence. The crRNA provides high-fidelity recognition through complementary base pairing, while the Cascade complex provides the catalytic and structural functions. This intermediary mechanism enables both high fidelity and adaptability to various target loci including difficult-to-access regions.
Solution Approach 2:
The system allows easy modification of target specificity by changing the crRNA sequence parameters. The crRNA length, composition, and sequence can be optimized for different target loci, enabling the system to adapt to difficult-to-access regions while maintaining high fidelity through controlled parameters such as guide sequence length and PAM sequence requirements.
Data Source
AI summary
A clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for adaptive antiviral defence (Cascade); the Cascade protein complex comprising at least CRISPR-associated protein subunits Cas7, Cas5 and Cas6 which includes at least one subunit with an additional amino acid sequence possessing nucleic acid or chromatin modifying, visualising, transcription activating or transcription repressing activity. The Cascade complex with additional activity is combined with an RNA molecule to produce a ribonucleoprotein complex. The RNA molecule is selected to have substantial complementarity to a target sequence. Targeted ribonucleoproteins can be used as genetic engineering tools for precise cutting of nucleic acids in homologous recombination, non-homologous end joining, gene modification, gene integration, mutation repair or for their visualisation, transcriptional activation or repression. A pair of ribonucleotides fused to FokI dimers may be used to generate double-strand breakages in the DNA to facilitate these applications in a sequence-specific manner.