Guide RNA Spacer Linker for CRISPR-Cas9 Targeting
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR-Cas9 systems face limitations in delivering specific RNA sequences to target nucleic acids with precision and efficiency, particularly in terms of RNA localization and functional integration with CRISPR complexes.
Innovation Solution
The method involves providing a Cas9 protein and a guide RNA with a selected RNA domain that forms a co-localization complex with the target nucleic acid, utilizing a spacer sequence, a tracr mate sequence, and a tracr sequence, which can be connected by a linker, to deliver the selected RNA sequence to the target site, allowing for precise RNA localization and functional interaction with the CRISPR system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a standard guide RNA structure is used in CRISPR-Cas9 systems, then the system can recognize and bind target DNA through PAM sequence probing, but the ability to deliver additional functional RNA sequences to the target site is limited
Solution Approach 1:
The patent combines the standard guide RNA structure with additional functional RNA sequences to create an extended guide RNA molecule. This merging allows the single RNA molecule to perform both target recognition (via the guide RNA portion) and functional delivery (via the attached RNA sequence), thereby increasing versatility without requiring separate molecular components
Solution Approach 2:
The extended guide RNA structure serves multiple functions: it maintains the ability to guide Cas9 to target DNA through conventional base pairing, while simultaneously delivering additional RNA sequences to the target site. This multi-functionality allows a single molecular construct to perform both navigation and cargo delivery roles
2Adaptability or versatility
If the guide RNA is extended to include additional functional sequences, then the functionality and versatility of CRISPR applications are expanded, but the precision and efficiency of RNA localization to the target site may be compromised
Solution Approach 1:
The patent applies local quality by maintaining the standard, highly precise guide RNA structure in the target-recognition region while attaching functional RNA sequences in a manner that does not interfere with the critical base-pairing region. This ensures that the localization precision is preserved in the essential region while gaining additional functionality elsewhere in the molecule
3Ease of manufacture
If separate crRNA and tracrRNA sequences are used, then the CRISPR system follows the natural bacterial mechanism, but the ease of engineering and customization of RNA sequences is reduced
Solution Approach 1:
The patent merges crRNA and tracrRNA into a single fused RNA molecule, which simplifies engineering and customization since only one RNA sequence needs to be designed and introduced. This unified structure maintains reliability by preserving the essential functional domains of both original components while enabling easier molecular biology manipulation
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 enables efficient and precise delivery of selected RNA sequences to target nucleic acids, enhancing the functionality of CRISPR-based methods by allowing for locus-specific targeting and interaction with RNA-guided binding proteins, thereby expanding the repertoire of CRISPR applications.
Implementation Method 1
after which Watson-Crick base-pairing between the gRNA and target DNA proceeds in a ratchet mechanism to form an R-loop
Data Source
AI summary
CRISPR/Cas Systems are provided where guide RNAs include one or more selected RNA sequences for delivery to a target nucleic acid sequence.