Modular Cas13d CRISPR Effectors for Diverse Nucleic Acid Editing
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Solution Overview
Problem
Current CRISPR-Cas systems are limited in their programmable effectors for nucleic acid modification, necessitating the development of additional systems with unique properties for diverse biotechnological applications.
Innovation Solution
Engineering non-naturally occurring CRISPR-Cas systems with accessory proteins containing WYL domains and CRISPR-associated proteins, such as Type VI-D CRISPR-Cas effector proteins, to modulate activities like nuclease activity, targeting specific nucleic acids, and incorporating base-editing domains for precise nucleic acid modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If current CRISPR-Cas systems are used for nucleic acid modification, then basic genome editing functions are achieved, but the diversity and versatility of programmable effectors are limited
Solution Approach 1:
The patent applies universality by designing a modular CRISPR system where a single Cas13d effector protein can perform multiple functions (RNA cleavage, base editing, transcriptional regulation) by simply changing the guide RNA sequence and accessory protein components. This multi-functional platform resolves the contradiction by providing diverse applications without requiring multiple different effector proteins, thus maintaining system simplicity while achieving versatility.
Solution Approach 2:
The patent employs parameter changes by modifying the chemical properties of nucleic acids through base editing (converting C to U, A to G, etc.) and by adjusting the composition of accessory proteins (WYL domain proteins, HEPN domain proteins) to change the effector's activity parameters. This allows the same core Cas13d system to achieve diverse nucleic acid modifications by changing molecular parameters rather than structural complexity.
2Manufacturing precision
If accessory proteins with WYL domains are added to modulate CRISPR-associated protein activity, then nuclease activity and targeting precision are enhanced, but system complexity increases
Solution Approach 1:
The patent uses WYL domain proteins as intermediary components that mediate between the guide RNA-Cas13d complex and the target nucleic acid. These accessory proteins enhance targeting precision by providing additional RNA-binding capabilities and structural stabilization, acting as mediators that improve precision without requiring fundamental changes to the core effector mechanism.
Solution Approach 2:
The patent applies segmentation by dividing the CRISPR system into modular functional domains: the Cas13d core effector, guide RNA, and separate accessory proteins (WYL domain proteins, HEPN domain proteins). This segmentation allows each component to be optimized independently for its specific function while maintaining overall system simplicity through standardized interfaces and assembly protocols.
3Manufacturing precision
If base-editing domains are incorporated for precise nucleic acid modifications, then editing accuracy is improved, but the complexity of the effector protein increases
Solution Approach 1:
The patent merges the base editing function with the RNA-guided targeting capability of Cas13d by fusing base editing domains (such as ADAR deaminase domains) to the effector protein. This merging creates a unified molecule that combines targeting precision with editing functionality, achieving high base editing accuracy while avoiding the need for separate positioning systems that would increase complexity.
Solution Approach 2:
The patent implements self-service by designing effector proteins where the Cas13d domain itself provides the positioning and recognition functions, while the fused base editing domain automatically performs the modification once positioned. The system is self-sufficient, requiring no additional separate components for positioning or catalysis, thus achieving high precision without proportional increases in overall system complexity.
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
Enhances the capability of CRISPR systems to perform targeted nucleic acid modifications, including cleavage, transcription, and translation regulation, with applications in genome editing and disease treatment.
Implementation Method 1
the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid
Implementation Method 2
the CRISPR-associated protein is capable of binding to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence
Implementation Method 3
the accessory protein modulates an activity of the CRISPR-associated protein, wherein the activity is a nuclease activity
Data Source
AI summary
The disclosure describes novel systems, methods, and compositions for the manipulation of nucleic acids in a targeted fashion. The disclosure describes non-naturally occurring, engineered CRISPR systems, components, and methods for targeted modification of a nucleic acid.