Cas12a tracrRNA Tethering for Higher Genome Editing Efficiency
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Solution Overview
Problem
Existing genome editing technologies, particularly those utilizing Cas12a systems, face inefficiencies in targeting and modifying specific DNA sequences, which can be enhanced by tethering functional RNA molecules to Cas12a crRNA for improved genome editing capabilities.
Innovation Solution
The method involves hybridizing a functional RNA molecule with a Cas12a tracrRNA and a crRNA extension to tether them, forming a Cas12a ribonucleoprotein complex, and using this complex to insert or modify nucleotide sequences at specific loci in target DNA, enhanced by the presence of Cas12a tracrRNA in molar excess.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Cas12a system is used for genome editing, then specific DNA sequences can be targeted, but editing efficiency is insufficient
Solution Approach 1:
The patent combines multiple functional RNA molecules (tracrRNA and crRNA with 3' extension) into a single ribonucleoprotein complex with Cas12a nuclease. This merging of RNA components enhances the editing efficiency and reliability of the Cas12a system by creating a more stable and effective complex for targeted genome editing.
Solution Approach 2:
The tracrRNA serves as an intermediary molecule that facilitates the interaction between the crRNA and Cas12a nuclease. By providing a bridge that enhances complex formation and stability, the tracrRNA mediates improved editing efficiency without requiring direct modification of the target DNA sequence.
2Device complexity
If tracrRNA is deleted from Cas12a system, then system simplification is achieved, but editing efficiency decreases
Solution Approach 1:
The patent changes the concentration parameter of tracrRNA by providing it in molar excess relative to Cas12a nuclease. This parameter change optimizes the formation of the ribonucleoprotein complex, ensuring that sufficient tracrRNA is available to enhance editing efficiency while maintaining system functionality.
3Adaptability or versatility
If crRNA 3' extension is added, then functional RNA tethering is enabled, but RNA molecule complexity increases
Solution Approach 1:
The crRNA is designed with a pre-added 3' extension sequence before complex formation with Cas12a and tracrRNA. This preliminary structural modification enables the crRNA to tether functional RNA molecules, providing versatility for enhanced editing capabilities while the extension sequence itself remains a defined, manageable structural element.
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 significantly enhances the genome editing efficiency by increasing the capability of Cas12a nuclease to modify target DNA sequences, allowing for precise and efficient insertion, deletion, or alteration of nucleotides in eukaryotic cells.
Implementation Method 1
hybridizing (a) a functional RNA molecule including a Cas12a tracrRNA and a functional RNA moiety; and (b) a Cas12a crRNA that includes a 3′ extension capable of hybridizing with a segment of the functional RNA molecule
Data Source
AI summary
This disclosure provides compositions that include a polynucleotide including or encoding a Cas12a tracrRNA, and methods for their use in sequence-specific genome editing, especially of eukaryotic genomic sequences. In particular, this disclosure provides Cas12a tracrRNA-containing compositions and methods for their use in Cas12a-mediated editing of a target sequence, wherein the editing efficiency is increased in comparison to controls lacking the Cas12a tracrRNA.