Engineered Cpf1 CRISPR Enzymes for Precise DNA and RNA Targeting
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Solution Overview
Problem
There is a need for alternative and robust systems and techniques to target nucleic acids with a wide array of applications, particularly for precise genome and epigenome editing, that are affordable, easy to set up, and scalable, and can target multiple positions within eukaryotic genomes.
Innovation Solution
The development of novel DNA or RNA-targeting systems using non-naturally occurring or engineered Type V CRISPR-Cas loci effector proteins, such as Cpf1, which form complexes with nucleic acid components to modify target loci, including introducing strand breaks, without requiring tracrRNA, and can be delivered via vectors or delivery systems like liposomes or viral vectors.
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 systems are complex, expensive, and difficult to scale for multiple positions
Solution Approach 1:
The patent uses CRISPR-Cas9 systems where a guide RNA (copy of the target sequence) directs the Cas9 enzyme to the genomic target, replacing complex protein-DNA recognition systems with a simpler RNA-mediated targeting approach that can be easily replicated for multiple targets
Solution Approach 2:
The CRISPR-Cas9 system provides a universal platform for genome editing across different organisms and target sequences, with the same Cas9 enzyme able to target any genomic location by simply changing the guide RNA sequence, eliminating the need for custom protein engineering for each target
2Productivity
If CRISPR-Cas9 systems are used for genome editing, then scalability and ease of setup improve, but off-target effects increase
Solution Approach 1:
The patent modifies key parameters of the CRISPR system including using high-fidelity Cas9 variants with altered PAM recognition requirements, optimizing guide RNA length and sequence composition, and adjusting cellular conditions to enhance specificity while maintaining editing efficiency across multiple targets
Solution Approach 2:
The introduction of a PAM recognition requirement acts as an intermediary safety check that prevents off-target binding, as the Cas9 enzyme must recognize both the guide RNA-complementary sequence and the specific PAM motif, adding an extra layer of specificity without reducing scalability
3Adaptability or versatility
If Type V CRISPR-Cas systems (Cpf1) are used instead of Cas9, then versatility and reduced off-target effects improve, but delivery complexity increases
Solution Approach 1:
The patent segments the CRISPR system into modular components (Cpf1 enzyme, crRNA, and PAM sequence requirements) that can be independently optimized and delivered, with the ability to target both DNA and RNA substrates using the same core enzyme through different guide RNA designs
Solution Approach 2:
The patent extracts and utilizes the unique features of Cpf1 (such as its T-rich PAM requirement and RNA-targeting capability) while removing limiting factors, creating a versatile system that can edit both DNA and RNA with reduced off-target effects through simplified delivery approaches
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
These systems enable precise and efficient modification of genomic or epigenomic sequences, allowing for genome editing and integration of DNA inserts into eukaryotic cells, including non-dividing cells, with reduced off-target effects and improved scalability and versatility.
Implementation Method 1
The CRISPR-Cas system loci has more than 50 gene families and there is no strictly universal genes indicating fast evolution and extreme diversity of loci architecture. So far, adopting a multi-pronged approach, there is comprehensive cas gene identification of about 395 profiles for 93 Cas proteins.
Implementation Method 2
can be delivered via vectors or delivery systems like liposomes or viral vectors
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 or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.