Cpf1 CRISPR Effector for Precise Multiplex Genome Editing
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Solution Overview
Problem
There is a need for affordable, easy-to-set-up, and scalable genome engineering technologies that can target multiple positions within the eukaryotic genome, leveraging novel strategies and molecular mechanisms beyond existing methods like designer zinc fingers and TALEs.
Innovation Solution
The development of CRISPR-Cas systems, specifically utilizing Cpf1 effector proteins, which form complexes with guide RNA to target and introduce staggered cuts at specific genomic loci, enabling precise genome editing without requiring tracrRNA, and allowing for multiplexed editing and integration of DNA inserts through NHEJ-based mechanisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR-Cas systems are used for genome editing, then precision and efficiency of targeting are improved, but system complexity and difficulty of setup increase
Solution Approach 1:
The CRISPR-Cas system is divided into separate functional modules: the Cas protein (effector), guide RNA (targeting component), and PAM sequence (recognition element). This segmentation allows independent optimization of each component and simplifies system assembly and setup procedures while maintaining high targeting precision through the coordinated function of these modular elements.
2Productivity
If multiple genome positions are targeted simultaneously, then productivity and versatility are improved, but off-target effects increase
Solution Approach 1:
The PAM sequence acts as an intermediary recognition element that enhances the specificity of Cas protein binding to target DNA. By requiring a specific PAM sequence adjacent to the target site, the system achieves higher discrimination between on-target and off-target sites, enabling safe multiplexed editing of multiple genome positions simultaneously.
3Adaptability or versatility
If novel CRISPR effector proteins are developed, then adaptability and targeting range are improved, but reliability and optimization requirements increase
Solution Approach 1:
The system achieves broad adaptability and expanded targeting range by varying key parameters including the Cas protein variant (different species origins), guide RNA sequence composition and length, and PAM sequence requirements. These parameter changes allow customization for different applications and target sites while maintaining reliable function through established optimization protocols for each parameter set.
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
This approach provides efficient and precise genome editing capabilities, facilitating targeted perturbations and integrations in various cell types, including non-dividing human cells, with reduced off-target effects and expanded targeting range, particularly suitable for biotechnological applications.
Implementation Method 1
the Cpf1 effector protein forms a complex with one or more nucleic acid components, wherein the composition comprises a single nucleic acid component which comprises a guide sequence linked to a direct repeat sequence
Implementation Method 2
the effector protein induces a modification of the target locus of interest, wherein the modification is the introduction of a strand break, and the strand break is a staggered cut
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.