Nucleic Acid Scaffold for Precise CRISPR Genome Editing
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Solution Overview
Problem
Current CRISPR-Cas systems for genome editing face limitations in precision and efficiency, particularly in targeting specific nucleic acid sequences without causing off-target effects, and in forming stable complexes for site-directed binding and cleavage.
Innovation Solution
The development of a nucleic acid scaffold (NASC) composition comprising engineered nucleic acid sequences that form a scaffold capable of binding nucleic acid binding proteins, specifically CRISPR-Cas9 and CRISPR-Cpf1 proteins, to facilitate precise targeting and cleavage of DNA sequences by forming a complex with two or more engineered nucleic acid components and associated proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-Cas systems are used for genome editing, then the ability to target and cleave DNA sequences is improved, but precision and off-target effects are worsened
Solution Approach 1:
The guide RNA is divided into multiple separate components (crRNA and tracrRNA) that must assemble together to form the functional complex. This segmentation allows for more precise control over target recognition, as each component can be independently optimized for specific binding affinity and specificity, thereby reducing off-target effects while maintaining genome editing capability
Solution Approach 2:
The patent introduces engineered nucleic acid sequences that form scaffold structures acting as intermediaries between the Cas protein and the target DNA. These scaffold structures provide a more stable and precise platform for target recognition, enhancing positioning accuracy and reducing erroneous binding to off-target sequences
2Productivity
If CRISPR-Cas systems are used for genome editing, then the ability to cleave DNA sequences is improved, but stability of nucleoprotein complexes is worsened
Solution Approach 1:
The patent merges the crRNA and tracrRNA into a unified scaffold structure that simultaneously provides both guide function and structural stability. This combined structure ensures the nucleoprotein complex remains stable throughout the genome editing process while maintaining efficient DNA cleavage capability
Solution Approach 2:
The engineered nucleic acid scaffold forms a composite structure with distinct functional regions that provide both stability and activity. The scaffold incorporates structured elements that stabilize protein binding while maintaining the flexibility needed for efficient DNA cleavage, creating a composite material that optimizes both stability and productivity
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
Enhances the precision and efficiency of genome editing by allowing for the formation of stable nucleoprotein complexes that can selectively bind and cleave specific DNA sequences, reducing off-target effects and improving the accuracy of site-directed nucleic acid modifications.
Implementation Method 1
The repeat nucleic acid sequence 1a and the repeat nucleic acid sequence 1aC are connected through hydrogen-bonded base pairs
Data Source
AI summary
The present disclosure provides engineered polynucleotide sequences that form scaffolds and nucleoprotein complexes comprising such engineered polynucleotide sequences that form scaffolds and nucleic acid binding proteins. Nucleic acid sequences encoding the engineered polynucleotide sequences that form scaffolds, as well as expression cassettes, vectors and cells comprising such polynucleotide sequences, are described. A variety of methods for making and using the engineered polynucleotide sequences that form scaffolds are also disclosed.