Cpf1 CRISPR Enzyme Systems 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, to facilitate systematic reverse engineering of genetic variations and advance synthetic biology and medical applications.
Innovation Solution
The development of engineered CRISPR-Cas systems utilizing Cpf1 effector proteins, guide RNAs, and vector systems that enable precise genome targeting and editing, including methods for delivering these components to various cell types for targeted genome modifications, such as introducing strand breaks and integrating DNA inserts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas systems are used for genome editing, then precise genome targeting is achieved, but the complexity of the system increases
Solution Approach 1:
The CRISPR-Cas system is divided into separate functional modules: guide RNA molecules that target specific genomic sequences and Cas effector proteins that perform the editing function. This segmentation allows independent optimization of each component and simplifies the overall system design while maintaining precise genome targeting capability
Solution Approach 2:
The CRISPR-Cas system employs universal guide RNA structures that can be programmed to recognize any target sequence, making the system broadly applicable across different genomic locations and cell types without requiring fundamental redesign, thus reducing complexity while preserving precision
2Adaptability or versatility
If multiple genome positions are targeted, then comprehensive genetic analysis is enabled, but the difficulty of setup and operation increases
Solution Approach 1:
The guide RNA molecules follow a universal design principle where changing only the spacer sequence allows targeting of any genomic position. This universality enables multiplexed targeting of multiple genome positions simultaneously without complicating the overall experimental setup, as the same Cas effector protein can work with multiple different guide RNAs
Solution Approach 2:
Guide RNAs are designed and synthesized in advance with predetermined spacer sequences that correspond to specific target sites. This preliminary preparation allows researchers to plan multiple targeting positions beforehand and simply mix and match pre-designed guide RNAs with the Cas effector, reducing operational complexity during actual genome editing experiments
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 provide efficient and precise genome editing capabilities, enabling targeted perturbation and editing of specific sites without deleterious effects, and are applicable to a wide range of cell types, including mammalian, plant, and non-mammalian eukaryotic cells, with potential applications in biotechnology and biologic product enhancement.
Implementation Method 1
the guide sequence is capable of hybridizing with a target sequence
Implementation Method 2
a Cpf1 effector protein... capable of forming a complex with the Cpfl effector protein... enabling targeted perturbation and editing of specific sites
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.