Modified Cascade Ribonucleoproteins for Negatively Supercoiled DNA Cleavage

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Solution Overview

Problem

There is an ongoing need for improved agents for sequence/site specific nucleic acid detection and/or cleavage in genetic engineering and genomic research, particularly in CRISPR/Cas systems, where existing methods like zinc finger nucleases require extensive protein engineering for each new DNA locus and lack specificity.

Innovation Solution

Development of Cascade complexes comprising CRISPR-associated protein subunits with additional amino acid sequences providing nucleic acid or chromatin modifying, visualising, transcription activating, or transcription repressing activity, combined with a guide RNA molecule for precise targeting and cleavage of nucleic acids, including supercoiled DNA.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If zinc finger nucleases are used for DNA cleavage, then DNA cutting capability is achieved, but extensive protein engineering is required for each new DNA locus and specificity is lacking

Engineering Contradiction:

Improveadaptability to different DNA lociVSAvoidprotein engineering complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The system divides the DNA targeting function into two separate components: a guide RNA molecule that provides locus-specific recognition and a Cas protein that provides the cleavage activity. This segmentation allows the guide RNA to be easily redesigned for different DNA loci without modifying the Cas protein, thereby reducing protein engineering complexity while maintaining adaptability to different DNA loci.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The guide RNA acts as an intermediary between the Cas protein and the target DNA sequence. It mediates the specific recognition of different DNA loci by base pairing with the target sequence, allowing the same Cas protein to be used across multiple loci by simply changing the guide RNA sequence, thus reducing the need for extensive protein engineering.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If existing CRISPR systems are used, then nucleic acid detection and cleavage capability is provided, but affinity and specificity for supercoiled DNA is insufficient

Engineering Contradiction:

Improvenucleic acid cleavage capabilityVSAvoidbinding specificity to supercoiled DNA

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The patent modifies specific local regions of the Cas protein, particularly the N-terminal domain, to enhance its ability to recognize and bind to the supercoiled structure of DNA. These localized modifications improve the overall reliability and specificity of supercoiled DNA cleavage without altering the entire protein structure.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent employs parameter changes by modifying amino acid sequences in the Cas protein to optimize binding affinity for supercoiled DNA. Specific mutations are introduced to alter the protein's interaction parameters with the supercoiled structure, thereby improving measurement precision and binding specificity.

Inventive Principle:

Principle #35Parameter changes

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

The Cascade complexes enable high-affinity binding and specific cleavage of negatively supercoiled DNA, enhancing the precision and efficiency of nucleic acid modification and detection, allowing for targeted gene regulation and locus-specific alterations.

Implementation Method 1

CRISPR RNA (crRNA) contains a spacer sequence complementary to a target nucleic acid sequence... the CRISPR complex binds to and cleaves the target nucleic acid in a sequence-specific manner

Methodology Applied

Scientific EffectCRISPR RNA-guided DNA recognition:

Implementation Method 2

crRNA contains a spacer sequence complementary to a target nucleic acid sequence... binds to and cleaves the target nucleic acid in a sequence-specific manner

Methodology Applied

Scientific EffectNucleic acid hybridization:

Implementation Method 3

Cascade induces bending of target DNA upon protospacer binding

Methodology Applied

Scientific EffectDNA bending:

Data Source

PatentUS20250327048A1Modified cascade ribonucleoproteins and uses thereof

Publication Date: 2025.10.23 CARIBOU BIOSCIENCES INC

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.