NmeCas9 ssDNA Cleavage via TracrRNA Extraction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR-Cas systems require tracrRNA and specific PAM motifs for efficient DNA targeting, limiting their ability to target single-stranded DNA and introducing off-target effects.
Innovation Solution
The Neisseria meningitidis Cas9 (NmeCas9) enzyme, which can efficiently cleave single-stranded DNA in an RNA-guided, tracrRNA-independent manner, using guide RNAs without tracrRNA sequences and with a PAM-independent mechanism, employing its HNH domain for specific cleavage sites measured from the 5' end of the RNA-paired region.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional CRISPR-Cas systems use tracrRNA and specific PAM motifs for DNA targeting, then DNA targeting efficiency is improved, but the ability to target single-stranded DNA is limited and off-target effects increase
Solution Approach 1:
The patent extracts and removes the tracrRNA component from the CRISPR-Cas system, creating a tracrRNA-independent Cas9 variant. This extraction allows the system to function without the conventional tracrRNA-PAM motif requirement, thereby expanding substrate range to include single-stranded DNA while maintaining targeting specificity through the guide RNA alone
Solution Approach 2:
The patent changes the operational parameters of Cas9 by modifying it to function without tracrRNA and without requiring specific PAM motifs. This parameter change enables the enzyme to recognize and cleave single-stranded DNA targets through alternative mechanisms, expanding the system's versatility while preserving its reliability through refined guide RNA design
2Measurement precision
If CRISPR-Cas systems require tracrRNA and specific PAM motifs, then targeting precision is improved, but device complexity increases
Solution Approach 1:
By removing the tracrRNA component entirely, the patent simplifies the RNA guide structure from a two-component system (crRNA-tracrRNA complex) to a single-component system. This extraction reduces device complexity while maintaining targeting precision through the optimized guide RNA sequence that directly guides Cas9 to the target without requiring PAM motifs
Solution Approach 2:
The guide RNA in this invention assumes multiple functions that were previously distributed between crRNA and tracrRNA. It provides both target recognition and Cas9 activation functions independently, eliminating the need for separate tracrRNA and PAM motifs, thus reducing complexity while preserving precision
3Productivity
If conventional CRISPR systems use dual RNA guidance, then DNA cleavage efficiency is improved, but the requirement for complex RNA guides increases
Solution Approach 1:
The patent merges the functions of crRNA and tracrRNA into a single guide RNA molecule that operates independently. This merged structure maintains the cleavage efficiency of dual-RNA systems by preserving essential functional elements while eliminating the need for separate tracrRNA, thereby reducing structural complexity without sacrificing productivity
Solution Approach 2:
The guide RNA in this system is self-sufficient and does not require tracrRNA for its function. It autonomously guides Cas9 to the target and facilitates cleavage efficiency through its own structural features, eliminating the need for the additional tracrRNA component and simplifying the overall system while maintaining high 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
NmeCas9 provides precise and efficient RNA-guided cleavage of single-stranded DNA, reducing off-target effects and expanding the range of targetable DNA substrates, including viruses like hepatitis B and HIV, with improved specificity and reduced requirement for complex RNA guides.
Implementation Method 1
NmeCas9 provides precise and efficient RNA-guided cleavage of single-stranded DNA
Data Source
AI summary
Many strains of the human pathogen Neisseria meningitidis carry a compact Cas9 (NmeCas9) that can serve to limit genetic exchange via natural transformation. Cas9 orthologues (including NmeCas9) have recently been adopted for RNA-guided genome engineering and DNA binding, adding to the need to define better their activities and properties. The present invention examines DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 is found able to cleave single-stranded DNA (ssDNA) targets in a manner that is RNA-guided but both PAM- and tracrRNA-independent. Beyond the requirement for guide-target pairing, this activity has no apparent sequence requirements, and the cleavage sites are measured from the 5′ end of the DNA substrate's RNA-paired region. These results indicate that tracrRNA domains are not strictly required for enzymatic activation of NmeCas9, and expand the list of targeting activities exhibited by these revolutionary RNA-guided nucleases.