Split-Nexus Cas9 Polynucleotides for DNA Binding Specificity
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Solution Overview
Problem
Current CRISPR-Cas9 systems face limitations in efficient target DNA recognition and cleavage, particularly with truncated single-chain crRNA/tracrRNA molecules, which affect binding and cleavage efficiency.
Innovation Solution
An engineered Type II CRISPR-Cas9 system utilizing three separate polynucleotides to form a complex with the Cas9 protein, where at least two polynucleotides form a nexus stem element, enhancing target DNA binding and cleavage specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If truncated single-chain crRNA/tracrRNA molecules are used, then the system complexity is reduced, but the binding and cleavage efficiency deteriorates
Solution Approach 1:
The guide RNA is divided into separate crRNA and tracrRNA components instead of using a single-chain molecule. This segmentation allows each component to maintain its specific structural requirements while working together, resolving the conflict between simplicity and efficiency by distributing functional requirements across multiple simpler components.
Solution Approach 2:
The invention modifies the structural parameters of the guide RNA by establishing specific stem element configurations (lower stem, upper stem, nexus stem) with defined lengths and base-pairing characteristics. These parameter changes optimize the binding and cleavage efficiency while maintaining system manageability through standardized structural elements.
2Manufacturing precision
If separate polynucleotides are used to form the CRISPR complex, then the manufacturing precision can be improved, but the device complexity increases
Solution Approach 1:
The guide RNA system is segmented into distinct polynucleotide components (crRNA and tracrRNA) with specific structural elements. This segmentation enables precise control over each component's sequence and structure, improving manufacturing precision for target DNA binding while organizing complexity into manageable, functionally-defined segments.
Solution Approach 2:
Different regions of the guide RNA components are assigned specific local qualities and functions: the lower stem region for structural stability, the upper stem for Cas9 interaction, and the nexus stem for cleavage activation. This local differentiation allows each region to be optimized independently for its specific function, improving overall precision without requiring uniform complexity throughout the entire system.
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
This approach improves the binding and cleavage efficiency of the Cas9 protein to specific DNA sequences, supporting next-generation CRISPR technologies with enhanced target recognition and activity.
Implementation Method 1
at least two of the three polynucleotides are necessary to form a nexus stem element
Implementation Method 2
The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner
Data Source
AI summary
The present specification discloses engineered Type II CRISPR-Cas9 systems comprising split-nexus Cas9-associated polynucleotides (sn-casPNs), including systems comprising three split-nexus Cas9-associated polynucleotides (sn1-casPN/sn2-casPN/sn3-casPN) and systems comprising two split-nexus Cas9-associated polynucleotides (sn1-casPN/sn2-casPN). Together with a Cas9 protein, the sn-casPNs facilitate site-specific modifications, including cleavage and mutagenesis, of a target polynucleotide in vitro and in vivo. Furthermore, the engineered Type II CRISPR-Cas9 systems comprising sn-casPNs are useful in methods of regulating expression of a target nucleic acid. Methods are described herein for the creation of a variety of engineered Type II CRISPR-Cas9 systems comprising two or more sn-casPNs. Polynucleotide sequences, expression cassettes, vectors, compositions, and kits for carrying out a variety of methods are also described. Furthermore, the present specification provides genetically modified cells, compositions of modified cells, transgenic organisms, pharmaceutical compositions, as well as a variety of compositions and methods involving the engineered Type II CRISPR-Cas9 systems.