Engineered Cas13 Proteins for Specific RNA Targeting
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Solution Overview
Problem
There is a pressing need for alternative and robust systems and techniques for targeting nucleic acids with diverse applications, particularly in developing effector proteins with altered functionality such as increased or decreased specificity, activity, and alternative PAM recognition, to effectively utilize RNA-targeting systems for genomic, transcriptomic, and epigenomic editing without deleterious effects.
Innovation Solution
Engineered CRISPR-Cas proteins with modified amino acids, specifically those containing HEPN domains and RxxxxH motifs, are developed to interact with guide RNA and form complexes, enhancing the specificity and activity of RNA-targeting systems like Type VI CRISPR-Cas13 proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Cas13 systems are used for RNA targeting, then RNA knockdown and transcript labeling are achieved, but non-specific RNA degradation occurs
Solution Approach 1:
The patent converts the harmful non-specific RNase activity of Cas13 into a beneficial signal by using collateral cleavage of a separate reporter RNA (like crRNA or a designed reporter) to indicate target detection. This allows the harmful activity to be harnessed for ultra-sensitive virus detection and RNA imaging applications.
Solution Approach 2:
The patent introduces intermediary elements such as structured RNA reporters and modified crRNAs that mediate between the Cas13 complex and the target RNA. These intermediaries allow specific target detection while controlling and directing the RNase activity away from non-specific degradation of cellular RNA.
2Adaptability or versatility
If CRISPR-Cas9 is used for genome editing, then DNA editing capability is achieved, but application to RNA targeting is limited
Solution Approach 1:
The patent discovers and characterizes multiple Cas13 variants (Cas13a, Cas13b, Cas13c, Cas13d) that can all be programmed with CRISPR RNA guides to target RNA, creating a universal RNA-targeting platform. Each variant has slightly different properties, allowing selection based on specific application needs while maintaining the core RNA targeting function.
Solution Approach 2:
The patent utilizes parameter changes by comparing and selecting different Cas13 variants with different kinetic properties, specificities, and PAM requirements. This allows optimization of the system for different applications - some variants may be better for detection, others for knockdown, depending on their inherent parameters.
3Measurement precision
If novel Cas13b orthologues are developed, then RNA-targeting specificity is improved, but extensive engineering and optimization is required
Solution Approach 1:
The patent segments the CRISPR system into modular components - the Cas13b orthologue protein, the CRISPR RNA guide, and optional PAM recognition elements. This modular segmentation allows independent optimization of each component and facilitates engineering of new variants with specific properties without redesigning the entire system.
Solution Approach 2:
The patent employs partial action by using truncated or modified versions of Cas13b proteins that retain core RNA-targeting function but have reduced complexity. This allows achieving sufficient specificity for many applications without requiring full-length, highly complex engineered variants.
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
The engineered CRISPR-Cas proteins improve the specificity and activity of RNA-targeting systems, enabling precise and efficient manipulation of nucleic acids, thereby transforming the study and perturbation of specific target sites through direct detection, analysis, and manipulation.
Implementation Method 1
interact with a guide RNA that forms a complex with the engineered CRISPR-Cas protein
Implementation Method 2
Cas13 binding to target single-stranded RNA activates a general RNase activity that cleaves the target and degrades surrounding RNA non-specifically
Data Source
AI summary
The present disclosure provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides mutated Cas13 proteins and their use in modifying target sequences as well as mutated Cas13 nucleic acid sequences and vectors encoding mutated Cas13 proteins and vector systems or CRISPR-Cas13 systems.