Engineered CRISPR Effector Proteins for Versatile DNA and RNA Targeting
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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 using novel strategies and molecular mechanisms, as existing methods like designer zinc fingers and TALEs are limited in versatility and efficiency.
Innovation Solution
Development of novel DNA- and RNA-targeting systems using engineered CRISPR-Cas systems, specifically Type V and VI CRISPR-Cas loci effector proteins, 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.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If designer zinc fingers or TALEs are used for genome editing, then targeted genome perturbations can be achieved, but the methods are limited in versatility and efficiency for targeting multiple positions
Solution Approach 1:
The CRISPR system segments the genome targeting function into modular components: a reusable Cas9 effector protein and interchangeable guide RNAs (gRNAs). Each gRNA contains a 20-nucleotide spacer sequence that can be customized to target any genomic location, allowing multiple target positions to be addressed by simply changing the gRNA sequence rather than redesigning the entire system.
Solution Approach 2:
The Cas9 effector protein serves as a universal platform that can recognize and bind to any DNA sequence when paired with an appropriate gRNA. This single effector protein can be used repeatedly across numerous different target sites by simply更换 the guide RNA, providing multi-functionality and high versatility for genome editing applications.
2Ease of manufacture
If existing genome-editing techniques are used, then targeted perturbations are possible, but they are not affordable, easy to set up, or scalable
Solution Approach 1:
The patent replaces complex protein-DNA recognition mechanisms (zinc fingers, TALEs) with a simpler RNA-DNA hybridization mechanism. The gRNA uses Watson-Crick base pairing to recognize target sequences, which is a more straightforward and easier-to-implement mechanism that can be achieved through standard molecular biology techniques like in vitro transcription and transfection.
Solution Approach 2:
The system allows easy modification of targeting parameters by simply changing the nucleotide sequence of the gRNA spacer region. This parameter change approach enables rapid adaptation to new target sites without requiring changes to the effector protein or other system components, greatly improving ease of setup and scalability.
3Manufacturing precision
If CRISPR-Cas systems are used for genome editing, then precise modification of genomic sequences is achieved, but off-target effects may occur
Solution Approach 1:
The system enhances local quality of target recognition by focusing the 20-nucleotide spacer sequence of the gRNA specifically on the unique target region. This localized specificity, combined with the requirement for a protospacer adjacent motif (PAM) sequence next to the target site, ensures that Cas9 only cuts at the intended location and not at similar but non-identical sequences, thereby reducing off-target effects.
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 and epigenomic sequences, facilitating genome editing and integration of DNA inserts in various cell types, including non-dividing cells, with reduced off-target effects and improved targeting versatility.
Implementation Method 1
a guide RNA, wherein the guide RNA comprises a spacer sequence and a direct repeat sequence
Implementation Method 2
the effector protein induces the modification of the sequences associated with or at the target locus of interest
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.