Engineered Cas13f Polypeptide Reduces Collateral RNA Cleavage
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Solution Overview
Problem
Existing CRISPR-Cas13 systems face challenges due to high collateral RNA cleavage activity, which can lead to cytotoxicity and limit their clinical application, particularly in eukaryotic cells.
Innovation Solution
Engineering a Cas13f polypeptide with specific mutations, such as Y666A and Y677A, to enhance spacer sequence-specific cleavage activity while reducing spacer sequence-independent collateral cleavage activity, thereby improving safety and efficacy in RNA targeting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-Cas13 systems are used for RNA targeting, then targeted RNA cleavage activity is achieved, but spacer sequence-independent collateral cleavage activity causes cytotoxicity
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (Y666A and Y677A) in the Cas13f polypeptide sequence. These mutations alter the biochemical parameters of the enzyme to reduce its collateral cleavage activity while preserving targeted RNA cleavage capability, directly resolving the technical contradiction between reliability and harmful effects
Solution Approach 2:
The patent applies local quality by making site-specific mutations at positions 666 and 677 in the Cas13f polypeptide. Instead of modifying the entire enzyme, localized changes are introduced at specific residues to differentially affect targeted versus collateral cleavage activities, thereby reducing cytotoxicity while maintaining therapeutic efficacy
2Object-affected harmful factors
If Cas13f polypeptide with mutations is engineered, then collateral cleavage activity is reduced, but cleavage activity may be compromised
Solution Approach 1:
The patent optimizes parameters by carefully selecting specific amino acid substitutions (Y666A and Y677A) that independently reduce collateral activity while experiments confirm they preserve or even enhance targeted cleavage activity, thus resolving the contradiction between reducing harm and maintaining efficacy
Solution Approach 2:
The patent employs feedback through systematic evaluation of mutant Cas13f variants, where cleavage activity and collateral effects are measured and used to guide further optimization. This iterative process ensures that mutations reducing collateral activity do not compromise targeted cleavage functionality
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 Cas13f polypeptide maintains or enhances targeted RNA cleavage activity while significantly reducing unintended RNA cleavage, making it suitable for therapeutic applications in eukaryotic cells with reduced toxicity.
Implementation Method 1
a spacer sequence capable of hybridizing to a target RNA, thereby guiding the complex to the target RNA
Implementation Method 2
the engineered Cas13f polypeptide... has an increased spacer sequence-specific cleavage activity
Data Source
AI summary
Provided are engineered Cas13f polypeptides, system or compositions comprising the same, and methods of using the same.