Modified Guide RNA Duplex for Specific Cas9 Target Recognition
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Solution Overview
Problem
Existing CRISPR-Cas systems face challenges in achieving efficient and specific site-directed DNA cleavage, nicking, transcriptional control, and genome editing due to limitations in the interaction between crRNA and tracrRNA, which affect target recognition and cleavage efficiency.
Innovation Solution
Modifying the crRNA:tracrRNA duplex by inserting, substituting, or deleting nucleotides or base pairs in the nexus hairpin and stem regions to form specific secondary structures, along with a linker, to enhance the interaction with Cas9 polypeptide, thereby improving the specificity and efficiency of site-specific cleavage, nicking, and genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the crRNA:tracrRNA duplex structure is modified by inserting, substituting, or deleting nucleotides in the nexus hairpin and stem regions, then the interaction with Cas9 polypeptide is enhanced and target recognition specificity is improved, but the complexity of the guide RNA construct increases
Solution Approach 1:
The patent applies local quality by making specific modifications only to certain regions of the crRNA:tracrRNA duplex (nexus hairpin and stem regions) while leaving other regions unchanged. This targeted approach enhances Cas9 interaction and target recognition specificity without unnecessarily complicating the entire construct, resolving the contradiction between reliability improvement and complexity increase.
Solution Approach 2:
The patent employs parameter changes by inserting, substituting, or deleting specific nucleotides (1-5 nucleotides in nexus hairpin, 1-9 nucleotides in stem) to optimize the secondary structure formation. These precise parameter modifications enhance the guide RNA's ability to interact with Cas9 and recognize targets specifically, achieving improved reliability with controlled complexity.
2Manufacturing precision
If modifications are made to enhance Cas9 activity and reduce off-target effects, then the precision of genome editing is improved, but the ease of manufacture of the guide RNA decreases
Solution Approach 1:
The patent applies preliminary action by pre-designing and pre-testing various nucleotide modifications in the nexus hairpin and stem regions to identify sequences that optimize Cas9 activity and reduce off-target effects. This preliminary optimization allows for standardized, easily manufacturable guide RNA constructs with high precision editing capabilities, resolving the contradiction between manufacturing precision and ease of manufacture.
3Productivity
If the nexus hairpin and stem regions are modified to form specific secondary structures, then the interaction efficiency with Cas9 is improved, but the time required for guide RNA design and optimization increases
Solution Approach 1:
The patent systematically varies nucleotide parameters (insertions, substitutions, deletions) in the nexus hairpin and stem regions to identify optimal secondary structures that maximize Cas9 interaction efficiency. By establishing specific parameter ranges (1-5 nucleotides in nexus, 1-9 in stem), the patent accelerates the design process while maintaining high productivity, resolving the contradiction between interaction efficiency and design time.
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 modified crRNA:tracrRNA constructs enhance the activity of Cas9 polypeptide, allowing precise control over DNA transcription, site-specific cleavage, and editing, with improved target recognition and reduced off-target effects.
Implementation Method 1
when the 5' region of the tracrRNA that is complementary to the 3' region (i.e., CRISPR repeat) of the crRNA hybridizes to the 3' region of the crRNA, the synthetic nucleic acid construct forms secondary structures from 5' to 3' of: (i) a stem, the stem comprising a duplex between the 5' end of the tracrRNA and the repeat of the crRNA
Implementation Method 2
at least a portion of the 5' region of the tracrRNA is complementary to the 3' region (i.e., CRISPR repeat) of the crRNA, wherein, when the 5' region of the tracrRNA that is complementary to the 3' region of the crRNA hybridizes to the 3' region of the crRNA
Implementation Method 3
The modified crRNA:tracrRNA constructs enhance the activity of Cas9 polypeptide, allowing precise control over DNA transcription, site-specific cleavage, and editing
Implementation Method 4
allowing precise control over DNA transcription, site-specific cleavage, and editing
Data Source
Figure 1A~1E
Figure 2A~2C
Figure 3
AI summary
The present invention is directed to modified CRISPR-cas guides that modulate the activity of Cas9 polypeptides to which the synthetic guides are complexed. In addition, methods of use of the modified CRISPR-cas guides are provided.