Segmented tracrRNA unit for CRISPR-Cas9 genome editing
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Solution Overview
Problem
The functionality of tracrRNA in the CRISPR-Cas9 system is unclear when its regions other than the linker segment between the first and second stem-loops are modified.
Innovation Solution
A novel tracrRNA unit comprising multiple short, non-continuous single-stranded RNAs that are complementary to each other, allowing for increased flexibility and functionality in genome editing by splitting the tracrRNA into segments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If the tracrRNA is split into multiple non-continuous single-stranded RNAs, then the synthesis cost is reduced and the number of functional motifs is increased, but the structural complexity and difficulty of maintaining functional integrity increase
Solution Approach 1:
The patent applies segmentation by dividing the traditional continuous tracrRNA into multiple separate single-stranded RNA components (first single-stranded RNA, second single-stranded RNA, and optionally third single-stranded RNA). Each segment contains specific functional motifs (such as stem-loop structures) that can independently interact with crRNA and Cas9. This segmentation reduces synthesis costs as shorter RNA fragments are cheaper to produce chemically, while the modular design allows for optimized functional motifs in each segment.
2Adaptability or versatility
If the tracrRNA is modified or partially deleted, then the synthesis cost is reduced and flexibility is increased, but the functional integrity and reliability may be compromised
Solution Approach 1:
By segmenting tracrRNA into separate components, the patent enables independent modification of each segment without affecting the entire molecule. Researchers can optimize individual segments for specific functions (e.g., enhancing crRNA binding in the first segment, improving Cas9 interaction in the second segment) while maintaining overall system functionality. This modular approach increases adaptability for different genome editing applications.
Solution Approach 2:
The patent design allows the segmented tracrRNA components to perform multiple functions. The first single-stranded RNA can bind to crRNA, the second segment can interact with Cas9, and additional segments can incorporate functional motifs for enhanced specificity, reduced off-target effects, or improved stability. This multi-functionality increases versatility while maintaining reliability through distributed functional elements.
3Adaptability or versatility
If the tracrRNA is divided into separate segments, then the number of functional motifs is increased and applications are enhanced, but the complexity of assembly and operation increases
Solution Approach 1:
The patent divides tracrRNA into separate single-stranded RNA segments that can be independently synthesized and then assembled. Each segment contains specific functional motifs (stem-loops, binding regions) that can be optimized for different purposes. The segments naturally assemble through complementary base pairing with crRNA and Cas9, reducing the need for complex assembly protocols despite the increased number of functional elements.
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 novel tracrRNA unit maintains its functional integrity even when partially deleted or modified, reducing synthesis costs and enhancing applications in genome editing by increasing the number of functional motifs.
Implementation Method 1
a CRISPR RNA (crRNA) having a nucleotide sequence complementary to a nucleotide sequence of a region of interest in genomic DNA and a tracrRNA having a nucleotide sequence complementary to the crRNA are separate RNA strands, which hybridize via the nucleotide sequences that are complementary to each other
Data Source
Figure 1(A)~1(C)
Figure 2
Figure 3a~3b
AI summary
An object of the present invention is to provide a novel tracrRNA unit and a genome editing method using this unit. The objects are achieved by a tracrRNA unit comprising a first single-stranded RNA and a second single-stranded RNA for use in a CRISPR-Cas9 genome editing system wherein the first single-stranded RNA and the second single-stranded RNA are not continuous, the first single-stranded RNA has at least a first segment and a second segment, the first and second segments do not overlap, the first segment has a sequence complementary to a part of a crRNA, and the second segment has a sequence complementary to the second single-stranded RNA.