Engineered CRISPR Effector Complexes for Multiplex Genome Targeting
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Solution Overview
Problem
Current genome-editing techniques lack affordability, ease of setup, scalability, and the ability to target multiple positions within eukaryotic genomes effectively, necessitating the development of novel strategies and molecular mechanisms for precise genome perturbation.
Innovation Solution
Employing non-naturally occurring CRISPR-Cas systems, specifically Type V and VI effector proteins, to form complexes with nucleic acid components for targeted genome editing, including Cpf1 and C2c1 loci proteins, which induce strand breaks at specific loci without requiring tracrRNA, and utilize engineered nucleic acid components for multiplexed targeting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional genome-editing techniques (zinc fingers, TALEs, homing meganucleases) are used, then targeted genome perturbations can be achieved, but the techniques are expensive, difficult to set up, and not scalable for multiple positions
Solution Approach 1:
The patent uses CRISPR-Cas systems where a guide RNA (a simple nucleic acid sequence) copies the targeting information from the desired genomic location, replacing complex protein-DNA recognition systems. The guide RNA serves as a programmable copy of the target sequence that directs the Cas enzyme to the correct location, dramatically simplifying the system while maintaining precision
Solution Approach 2:
The patent changes the fundamental parameter of genome editing from protein-based recognition (zinc fingers, TALEs) to RNA-based recognition (CRISPR guide RNA). This parameter change allows for easier design and scaling because RNA sequences can be rapidly synthesized and modified without complex protein engineering, enabling multiplexed targeting of multiple genomic positions simultaneously
2Ease of operation
If CRISPR-Cas systems are used for genome editing, then ease of setup and scalability improve, but off-target effects increase
Solution Approach 1:
The patent replaces the mechanical/protein-based recognition system with a nucleic acid-based recognition system. The guide RNA uses Watson-Crick base pairing to recognize target sequences, which is a more specific and programmable mechanism than protein-DNA interactions. This substitution reduces off-target effects while maintaining ease of setup
Solution Approach 2:
The patent introduces guide RNA as an intermediary between the Cas enzyme and the target DNA. This intermediary provides programmable specificity through its sequence, allowing precise targeting while the Cas enzyme provides the catalytic activity. The intermediary mechanism enables easy reprogramming by simply changing the guide RNA sequence without modifying the enzyme itself
3Device complexity
If single-subunit effector modules (Cas9) are used, then device complexity is reduced, but the ability to target multiple positions simultaneously is limited
Solution Approach 1:
The patent demonstrates that the CRISPR-Cas system with guide RNA achieves universality and multi-functionality. A single Cas enzyme can be programmed to target multiple different genomic positions by simply changing the guide RNA sequence. This allows one enzyme to perform multiple functions (targeting different loci) without requiring multiple different enzymes or complex systems
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 and precise genome editing in various cell types, including eukaryotic cells, with reduced off-target effects, facilitating applications in biotechnology and medicine by allowing selective perturbation of genetic elements.
Implementation Method 1
the effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of the sequences associated with or at the target locus of interest
Data Source
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AI summary
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. Methods for making and using and uses of such systems, methods, and compositions and products from such methods and uses are also disclosed and claimed.