CRISPR Effector Proteins for Targeted Nucleic Acid Detection and Editing
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Solution Overview
Problem
Current CRISPR/Cas systems face limitations in efficient in vitro detection and effective in vivo engineering for modifying target nucleic acids associated with diseases or disorders, necessitating the development of alternative strategies and components.
Innovation Solution
The use of effector proteins with engineered guide nucleic acids to modify, detect, and engineer target nucleic acids through cis and trans cleavage activities, including nucleic acid cleavage, silencing, and splicing, with variants of these proteins fused to fusion partners for targeted nucleic acid modification and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional CRISPR/Cas systems are used, then nucleic acid cleavage activity is achieved, but efficiency in vitro detection and effectiveness in vivo engineering are limited
Solution Approach 1:
The patent modifies the CRISPR/Cas system by changing key parameters including using alternative effector proteins (not limited to Cas proteins), engineering guide nucleic acids with specific sequences and structures, and optimizing reaction conditions to enhance both in vitro detection efficiency and in vivo engineering effectiveness while maintaining reliability
Solution Approach 2:
The effector proteins and guide nucleic acid complexes are designed to perform multiple functions: they can detect target nucleic acids through cleavage activity, modify nucleic acid sequences, and operate effectively both in vitro and in vivo, thereby resolving the contradiction between detection efficiency and engineering effectiveness
2Productivity
If effector proteins with guide nucleic acids are used, then target nucleic acid modification is achieved, but system complexity increases
Solution Approach 1:
The system is segmented into distinct functional components: effector proteins (which may be catalytically active or inactive), guide nucleic acids (which provide target recognition), and fusion partners (which provide modification functionality). This segmentation allows each component to be optimized independently while working together to achieve efficient nucleic acid modification
Solution Approach 2:
Guide nucleic acids serve as intermediaries that bridge the effector proteins and target nucleic acids, enabling specific recognition and modification. The guide nucleic acids facilitate the interaction between the effector proteins and their targets without being permanently modified, simplifying the overall system architecture
3Ease of operation
If catalytically inactive effector protein variants are fused to fusion partners, then targeted modification is achieved, but protein complexity increases
Solution Approach 1:
The catalytic activity is extracted from the effector protein and transferred to the fusion partner. The effector protein is designed as a catalytically inactive variant that retains only the target recognition and binding functions, while the fusion partner provides the modification capability. This extraction simplifies the effector protein structure and allows for more precise targeted modification
Solution Approach 2:
The functionality of multiple proteins is merged into a single fusion protein construct. The fusion partner combines the effector protein's target recognition domain with additional domains that provide catalytic activity and modification functions, creating a unified protein that achieves targeted modification while managing structural complexity through functional integration
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
Enables efficient detection and modification of target nucleic acids, facilitating the diagnosis and treatment of diseases or disorders associated with mutations, with enhanced specificity and efficacy.
Implementation Method 1
CRISPR/Cas systems provide immunity in bacteria and archaea against viruses and plasmids by targeting the nucleic acids of the viruses and plasmids in a sequence-specific manner
Implementation Method 2
effector proteins described herein exhibit nucleic acid cleavage activity. In some embodiments, nucleic acid cleavage activity comprises cis cleavage activity, trans cleavage activity, nicking activity, and/or nuclease activity
Implementation Method 3
The discovery of CRISPR/Cas systems has revolutionized the field of genomic manipulation and engineering. Yet, the discovery suffers from several shortcomings that restricts its use for basic biomedical research and therapeutic applications
Data Source
AI summary
Provided herein are compositions, systems, devices, kits, and methods comprising effector proteins, and uses thereof. These effector proteins may be characterized as CRISPR-associated (Cas) proteins. Various compositions, systems, devices, kits, and methods of the present disclosure may leverage the activities of these effector proteins for the modifying, detecting and/or engineering of nucleic acids.