Engineered CRISPR Enzymes With Guide RNAs for Multiplex Targeting
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Solution Overview
Problem
Current genome-editing technologies, such as designer zinc fingers and TALEs, are limited in their ability to target multiple positions within the eukaryotic genome efficiently and affordably, necessitating the development of novel strategies for precise genome perturbation and editing.
Innovation Solution
Employing engineered CRISPR-Cas systems, particularly Type V and VI CRISPR-Cas loci effector proteins like Cpf1, to form complexes with nucleic acid components for targeted modification of genomic and epigenomic loci, including the use of C2c1 and FnCpf1 proteins for precise DNA cleavage and integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional genome-editing technologies like designer zinc fingers and TALEs are used, then precise targeting of individual genetic elements is achieved, but the ability to efficiently target multiple positions within the eukaryotic genome is limited and costs increase
Solution Approach 1:
The CRISPR-Cas9 system employs a universal guide RNA structure that can direct the Cas9 endonuclease to multiple different genomic positions by simply changing the spacer sequence. This single system architecture serves multiple targeting functions, eliminating the need for different protein complexes for each target site, thereby achieving multi-position targeting efficiency while maintaining system simplicity
Solution Approach 2:
The guide RNA is segmented into distinct functional domains including the spacer sequence (for target recognition), the crRNA repeat (for Cas9 binding), and the tracrRNA (for processing and stability). This segmentation allows independent optimization of each component and enables modular design where only the spacer needs to be changed to target different positions, enhancing versatility without increasing overall system complexity
2Productivity
If traditional genome-editing technologies are used, then targeted genome perturbations are achieved, but affordability and scalability are reduced
Solution Approach 1:
The system uses RNA copies (guide RNA) to direct the editing function rather than requiring multiple unique protein complexes. The guide RNA can be easily synthesized in vitro and introduced into cells, providing a low-cost, scalable method for genome editing that avoids the complex and expensive production processes required for traditional protein-based editing tools
Solution Approach 2:
The CRISPR array in the bacterial system performs preliminary action by capturing and storing viral DNA sequences as spacers during prior infections. This pre-acquired library of spacers can be directly used to target multiple positions without requiring de novo design and testing of each guide sequence, thereby accelerating the editing process and reducing setup costs
3Adaptability or versatility
If CRISPR-Cas systems are used for genome editing, then multiplexed targeting capability is enhanced, but off-target effects may increase
Solution Approach 1:
The system employs multiple guide RNAs simultaneously (partial action on multiple targets) to achieve multiplexed editing. Each guide RNA targets a specific position with high specificity due to the requirement for exact complementarity between the spacer and the target sequence, including the PAM motif recognition. This partial targeting approach with multiple guides reduces off-target effects compared to using a single guide with lower specificity
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 scalable genome editing with reduced off-target effects, allowing for multiplexed targeting and integration of DNA inserts in various cell types, including non-dividing human cells, with enhanced specificity and control over cleavage mechanisms.
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
Implementation Method 2
the effector protein induces the modification of the sequences associated with or at the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break
Data Source
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.