CRISPR RNA Constructs with Modular Regions for Cas9 Targeting

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Solution Overview

Problem

Current synthetic type II CRISPR-Cas systems face limitations in efficiency and specificity for genome editing and other applications, requiring improvements to enhance their performance.

Innovation Solution

The development of synthetic trans-encoded CRISPR(tracr) and CRISPR nucleic acid constructs with specific sequences and structures, including anti-zipper, bulge, anti-stitch, nexus, and hairpin sequences, that interact with Cas9 nuclease to facilitate site-specific cleavage of target DNA by hybridizing with complementary regions and adjacent PAM motifs.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If current synthetic type II CRISPR-Cas systems are used for genome editing, then basic targeting function is achieved, but efficiency and specificity are limited

Engineering Contradiction:

Improveefficiency of site-specific cleavageVSAvoidspecificity of target recognition

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies local quality by designing specific structural regions within the CRISPR RNA molecule (zipper region, bulge region, stitch region, nexus region, and hairpin region) with distinct sequence and structural characteristics. Each region serves a specialized function: the zipper region mediates hybridization with the target, the bulge region provides structural flexibility, the stitch region stabilizes the complex, the nexus region connects functional elements, and the hairpin region enhances stability. This localized optimization of different regions resolves the contradiction by improving cleavage efficiency through specialized structural features while maintaining high specificity through precise sequence complementarity in the target-binding region.

Inventive Principle:

Principle #3Local quality

2Reliability

If CRISPR constructs with enhanced targeting capability are designed, then specificity improves, but system complexity increases

Engineering Contradiction:

Improvespecificity of target recognitionVSAvoidcomplexity of nucleic acid construct

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent applies segmentation by dividing the CRISPR RNA construct into distinct functional modules: zipper region (for target hybridization), bulge region (for structural flexibility), stitch region (for complex stabilization), nexus region (for connecting functional elements), and hairpin region (for enhanced stability). This modular segmentation allows each region to be independently optimized for its specific function while maintaining overall system coherence. The segmented design achieves high specificity through specialized regions without requiring uniformly complex construction throughout the entire molecule, thus resolving the contradiction between specificity and overall system complexity.

Inventive Principle:

Principle #1Segmentation

3Manufacturing precision

If synthetic CRISPR constructs are used for precise genome editing, then editing accuracy improves, but cleavage efficiency remains limited

Engineering Contradiction:

Improveprecision of genome editingVSAvoidefficiency of site-specific cleavage

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent applies preliminary action by incorporating pre-formed structural elements (bulge region, hairpin region, and stabilized stitch region) into the CRISPR construct before target recognition. These pre-configured structural features prepare the molecule for optimal target binding and Cas9 recruitment. The hairpin region, in particular, is pre-formed to enhance stability and facilitate efficient target hybridization. This preliminary structural organization enables the system to achieve both high precision editing (through accurate target recognition) and high cleavage efficiency (through pre-optimized structural readiness for Cas9 activation).

Inventive Principle:

Principle #10Preliminary action

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

These constructs significantly enhance the efficiency and specificity of site-specific cleavage and targeting of DNA, allowing for precise genome editing and polypeptide placement, as demonstrated by their ability to hybridize and cleave target sequences with high accuracy.

Implementation Method 1

The CRISPR RNA is processed into a mature complex that remains associated with the both the tracrRNA and Cas9. The mature complex then locates a target dsDNA sequence ('protospacer' sequence) that is complementary to the spacer sequence in the complex

Methodology Applied

Scientific EffectHybridization: