Type VI-E/F CRISPR-Cas Proteins for Compact RNA Targeting
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Solution Overview
Problem
Current CRISPR-Cas systems, particularly Class 2 type VI proteins like Cas13a, Cas13b, and Cas13d, are too large for efficient packaging into small gene therapy vectors and exhibit non-specific/collateral RNase activity, posing risks for gene therapy applications.
Innovation Solution
Development of smaller Class 2, type VI Cas proteins, such as Cas13e and Cas13f, which can be packaged into small vectors like AAV and exhibit minimal non-specific RNase activity, with specific RNA targeting capabilities and RNA editing functions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If Class 2 type VI Cas proteins (Cas13a, Cas13b, Cas13d) are used for RNA targeting, then RNA editing function is achieved, but the protein size is too large for efficient packaging into small gene therapy vectors
Solution Approach 1:
The patent extracts and utilizes only the essential catalytic domains (HEPN domains) of the Cas13 proteins, removing non-essential regions to create smaller functional variants (Cas13e, Cas13f) that retain RNA editing capability while reducing size for vector packaging
Solution Approach 2:
The patent modifies protein size parameters by creating truncated or engineered versions of Cas13 proteins that maintain functional activity while fitting within the size constraints of small gene therapy vectors like AAV
2Adaptability or versatility
If Class 2 type VI Cas proteins are used for RNA targeting, then RNA editing function is achieved, but non-specific/collateral RNase activity poses risks for gene therapy applications
Solution Approach 1:
The patent introduces point mutations in specific HEPN domains to locally modify the catalytic activity, creating variants with reduced collateral RNase activity while preserving on-target RNA editing function through careful domain engineering
Solution Approach 2:
The patent changes the catalytic parameters of the RNase activity by introducing mutations that reduce non-specific cleavage while maintaining specificity for guide RNA-directed targets, thereby improving safety profile
3Productivity
If smaller Cas13e and Cas13f proteins are engineered, then packaging efficiency into small vectors is improved, but protein engineering complexity increases
Solution Approach 1:
The patent segments the Cas13 protein into functional modules (HEPN domains, linker regions, C-terminal domains) that can be independently engineered and recombined to create optimized smaller variants with desired properties
Solution Approach 2:
The engineered Cas13e and Cas13f proteins maintain multiple functions (RNA binding, catalysis, guide RNA interaction) within a reduced size framework, achieving multi-functionality despite size reduction
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
Cas13e and Cas13f proteins efficiently target and edit RNA with high specificity, reducing off-target effects and enabling effective gene therapy applications.
Implementation Method 1
an RNA guide sequence comprising a spacer sequence capable of hybridizing to a target RNA
Implementation Method 2
a CRISPR-associated protein (Cas) having an amino acid sequence of any one of SEQ ID NOs: 1-7, or a derivative or functional fragment of said Cas; wherein the Cas, the derivative, and the functional fragment of said Cas, are capable of (i) binding to the RNA guide sequence and (ii) targeting the target RNA
Data Source
AI summary
The invention provides novel CRISPR/Cas compositions and uses thereof for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting Cas13e or Cas13f effector protein, and at least one targeting nucleic acid component such as a guide RNA (gRNA) or crRNA. The novel Cas effector proteins are among the smallest of the known Cas effector proteins, at about 800 amino acids in size, and are thus uniquely suitable for delivery using vectors of small capacity, such as an AAV vector.