RNA-Guided CRISPR Effectors for Specific RNA Targeting
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Solution Overview
Problem
Existing RNA-targeting CRISPR tools face challenges such as weak activity in mammalian cells, toxicity, and size limitations, hindering effective gene and cell therapies, particularly in delivering large payloads like single-vector homology directed repair and CRISPR base editing.
Innovation Solution
Development of RNA-guided CRISPR effectors, including guide RNAs and polypeptides with specific amino acid sequences, capable of cleaving RNA targets in cells, and nucleic acid molecules encoding these components for therapeutic and diagnostic applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-Cas systems are used for RNA targeting, then RNA binding and modulation capability is improved, but off-target effects and lack of transcriptional activation occur
Solution Approach 1:
The patent introduces distinct functional domains within the Cas13a effector structure, where different regions are optimized for specific functions: the catalytic domain for on-target RNA cleavage, and regulatory domains for controlling off-target effects. This local functional differentiation allows the system to achieve high specificity for intended targets while minimizing unwanted interactions with non-target RNAs.
Solution Approach 2:
The patent employs guide RNA molecules as intermediaries that mediate between the Cas13a effector and target RNA. The guide RNA provides sequence-specific recognition through base pairing, acting as a selective intermediary that directs the effector to intended targets while preventing indiscriminate binding to off-target RNAs. This intermediary layer enhances specificity without requiring modifications to the effector itself.
2Measurement precision
If CRISPR-Cas systems are designed for high specificity, then target recognition accuracy is improved, but transcriptional activation and gene expression modulation are limited
Solution Approach 1:
The patent engineers Cas13a effectors with multi-functional capabilities that enable both high-specificity target recognition and transcriptional activation. By fusing the Cas13a RNA-guided recognition domain with transcriptional regulator domains, the system achieves dual functionality: maintaining accurate target identification through guide RNA-complementarity while simultaneously activating or repressing gene expression at the transcriptional level.
Solution Approach 2:
The patent creates composite CRISPR systems by combining Cas13a protein domains with separate transcriptional activation domains in a single functional complex. This composite architecture allows the system to integrate the high-specificity RNA binding capability of Cas13a with the gene expression modulation capability of transcriptional regulators, achieving both precise target recognition and effective transcriptional control in one unified system.
3Productivity
If CRISPR components are optimized for RNA binding, then RNA-targeting efficiency is improved, but protein complexity and system design difficulty increase
Solution Approach 1:
The patent divides the CRISPR system into modular functional segments: the Cas13a effector protein, guide RNA molecules, and optional transcriptional regulator domains. Each segment can be independently optimized and characterized, then assembled into functional complexes. This segmentation allows researchers to optimize RNA-binding efficiency in the Cas13a domain while separately managing the complexity of transcriptional activation functions, reducing overall system design difficulty.
Solution Approach 2:
The patent employs dynamic regulatory elements that can be adjusted or removed based on specific application requirements. The system allows for flexible configuration where transcriptional activation domains can be added or omitted depending on whether gene expression modulation is needed, and guide RNA sequences can be dynamically changed to target different RNAs. This dynamic adaptability simplifies system design by allowing users to configure only the functional elements needed for their specific application.
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 RNA-guided CRISPR effectors efficiently cleave RNA targets, stabilize RNA, modulate translation, and treat genetically inherited diseases, while providing diagnostic capabilities through RNA tracking and splicing modulation.
Implementation Method 1
the guide RNA is complementary to a target sequence of the target RNA
Implementation Method 2
RNA-guided RNA-targeting CRISPR effectors and methods of use thereof. In one aspect, the present disclosure provides a method of identifying a target RNA. The method includes incubating a CRISPR effector protein with a guide RNA under conditions that allow binding of the CRISPR effector protein to the guide RNA
Implementation Method 3
the present application provides a method of modulating gene expression in a cell. The method includes introducing a polypeptide encoding a RNA-guided RNA-targeting CRISPR effector into the cell
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 1E
AI summary
This disclosure provides systems, methods, and compositions for RNA-guided RNA- targeting CRISPR effectors for the treatment of diseases as well as diagnostics. In some embodiments, nucleotide deaminase functionalized CRISPR systems for RNA editing RNA knockdown, viral resistance, splicing modulation, RNA tracking, translation modulation, and epi-transcriptomic modifications are disclosed.