Engineered CRISPR-Cas Enzymes for Precise Multiplex Targeting
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Solution Overview
Problem
Current genome editing technologies 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 engineered CRISPR-Cas systems, particularly Type V and VI CRISPR-Cas loci effector proteins like Cpf1, which form complexes with nucleic acid components to induce targeted modifications, such as strand breaks, at specific genomic or epigenomic loci without requiring tracrRNA, and utilizing optimized guide RNAs for precise targeting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional genome editing technologies (designer zinc fingers, TALEs, homing meganucleases) are used, then targeted genome perturbations can be achieved, but the systems are expensive, complex to set up, and difficult to scale
Solution Approach 1:
The patent employs CRISPR-Cas systems that use RNA guides as templates to direct nucleases to specific genomic locations, replacing complex protein-DNA recognition systems with simpler RNA-mediated targeting. The guide RNA sequences are designed to complement target DNA sequences, enabling precise genome editing without the complexity of traditional methods.
Solution Approach 2:
The invention replaces the mechanical and structurally complex zinc finger or TALE protein domains with a CRISPR-Cas nuclease guided by RNA. This substitution simplifies the system by using sequence-complementary RNA guides instead of complex protein structures that must be individually engineered for each target site.
2Measurement precision
If traditional genome editing technologies are used, then genome perturbation can be achieved, but affordability and ease of setup are compromised
Solution Approach 1:
The CRISPR system is divided into separate functional components: a nuclease (Cas9 or Cpf1) and guide RNAs. This segmentation allows the nuclease to be expressed once while multiple different guide RNAs can direct it to various target sites, simplifying setup and reducing costs compared to traditional methods that require entirely different proteins for each target.
Solution Approach 2:
The patent demonstrates that a single CRISPR-Cas nuclease can perform multiple genome editing functions at different genomic locations by simply changing the guide RNA sequence. This universal platform enables the same enzyme to target multiple positions within the eukaryotic genome, making the system both affordable and easy to set up.
3Measurement precision
If traditional genome editing technologies are used, then single position targeting is achievable, but scalability to multiple positions is limited
Solution Approach 1:
The CRISPR system enables multiplexed genome editing by allowing simultaneous expression of multiple guide RNAs that direct the same nuclease to different target sites. The patent describes systems where multiple spacers in a single CRISPR array can guide the nuclease to multiple positions within the genome, achieving both specificity and scalability.
Solution Approach 2:
The invention combines multiple targeting capabilities into a single CRISPR-Cas system by integrating multiple guide RNA sequences. This merging allows the system to simultaneously or sequentially edit multiple genomic positions, providing versatility while maintaining the simplicity and precision of the core nuclease mechanism.
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 with reduced off-target effects, facilitating applications in biotechnology and medicine by allowing selective perturbation of genetic elements and scalable editing across various cell types, including eukaryotic cells.
Implementation Method 1
The CRISPR-Cas system employs a guide RNA molecule that base-pairs with a complementary DNA sequence to direct the effector protein to a specific target locus
Implementation Method 2
The effector protein is capable of inducing a double-strand break at the target locus
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.