Engineered Cas13f HEPN Mutations for Collateral RNA Control
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Solution Overview
Problem
Current CRISPR-Cas13 proteins exhibit non-specific/collateral RNase activity upon target sequence recognition, which poses a barrier for in vivo applications like gene therapy due to undesirable collateral degradation of bystander RNAs, and there is a need to optimize them for either reducing or enhancing this activity based on the application.
Innovation Solution
Engineered Cas13f polypeptides with mutations in specific regions, such as near the N- or C-terminal endonuclease catalytic motifs, to preserve target-specific cleavage activity while significantly reducing or enhancing collateral cleavage activity, depending on the desired application.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If wild type Cas13f is used for RNA editing, then target-specific cleavage activity is achieved, but collateral RNase activity causes degradation of bystander RNAs
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (e.g., D160A, D642A) in the HEPN domains of Cas13f to alter the enzyme's catalytic properties. These mutations reduce the collateral RNase activity while preserving target-specific cleavage capability, effectively tuning the parameter of RNase activity from a harmful broad-spectrum function to a controlled specific function.
Solution Approach 2:
The invention applies local quality by making targeted mutations in specific regions (HEPN1 and HEPN2 domains) of the Cas13f protein while leaving the rest of the structure intact. The mutations are localized to residues spatially close to the catalytic RXXXXH motifs, allowing differential control of catalytic activity - reducing collateral damage while maintaining on-target efficiency.
2Reliability
If Cas13f mutations are introduced to reduce collateral activity, then in vivo therapeutic safety is improved, but detection sensitivity may be reduced
Solution Approach 1:
The patent applies dynamics by creating a family of Cas13f variants with different mutation combinations (e.g., single mutations like D160A, double mutations like D160A/D642A, and triple mutations) that provide a gradient of collateral activity reduction. This dynamic range allows selection of appropriate variants for different applications - more conservative mutations for therapy, less conservative for detection.
Solution Approach 2:
The invention segments the Cas13f protein into functional domains (HEPN1, HEPN2, IDL, Helical1, Helical2) and applies mutations selectively in the HEPN domains that are spatially close to catalytic motifs. This segmentation allows independent optimization of different functional aspects - catalytic activity control in HEPN domains while preserving target recognition capability in other domains.
3Measurement precision
If Cas13f mutations are introduced to enhance collateral activity, then diagnostic detection sensitivity is improved, but off-target RNA degradation increases
Solution Approach 1:
The patent applies copying by creating multiple mutant variants of Cas13f (e.g., F7V2, F10V1, F10V4, F40V4, F40S22, F40S26, F40S36) that replicate the wild type's target recognition capability while amplifying collateral activity for detection purposes. These copies are then selected and optimized for specific diagnostic applications where enhanced collateral signaling is beneficial.
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 polypeptides maintain high target-specific cleavage efficiency while minimizing collateral RNA degradation, making them suitable for therapeutic and diagnostic applications, respectively, and provide enhanced sensitivity in nucleic acid detection assays.
Implementation Method 1
CRISPR-Cas13 is quickly becoming a widely adopted RNA editing technology. This system can use its sequence specific guide RNA to selectively modify (e.g., cut or cleave via endonuclease activity) a target RNA, such as mRNA.
Implementation Method 2
This system can use its sequence specific guide RNA to selectively modify (e.g., cut or cleave via endonuclease activity) a target RNA
Data Source
AI summary
The disclosure provides novel engineered Cas13f effector proteins that substantially maintain guide sequence-specific cleavage activity and substantially lack guide sequence-independent collateral cleavage activity and uses thereof, such as in RNA-based target gene transcript knock down.