dCas13 Steric Blocking for Precise RNA Regulatory Control
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Solution Overview
Problem
Existing RNA modulation technologies, such as antisense oligonucleotides and CRISPR-Cas13 systems, suffer from off-target effects and inadequate target engagement, limiting their clinical application in modulating regulatory elements in nucleic acids.
Innovation Solution
Utilizing a catalytically inactive Cas13 protein (dCas13) recruited by a CRISPR RNA (crRNA) as a steric blocker to modulate regulatory elements in nucleic acids, specifically employing Psp-dCas13b with a 18-24 nucleotide crRNA spacer and C-terminal truncation for enhanced blocking efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antisense oligonucleotides or CRISPR-Cas13 systems are used to modulate RNA function, then target RNA can be degraded or edited, but off-target effects and inadequate target engagement occur
Solution Approach 1:
The invention extracts and removes the catalytic function from the Cas13 system by using a catalytically inactive mutant (dCas13), separating the RNA binding capability from the RNA degradation function. This allows the system to bind specifically to target RNA without causing off-target degradation effects.
Solution Approach 2:
The invention introduces dCas13 as an intermediary steric blocker that physically occupies the target site without degrading the RNA. This intermediary prevents other molecules from accessing the target site, achieving modulation through physical blockage rather than chemical degradation.
2Productivity
If catalytically active Cas13 is used to degrade target RNA, then RNA manipulation is achieved, but the target RNA sequence is altered and off-target effects occur
Solution Approach 1:
The catalytic activity responsible for RNA degradation is extracted from the Cas13 system through site-directed mutagenesis, creating dCas13 that retains RNA binding ability but lacks degradation function. This allows RNA manipulation through steric blocking without altering RNA sequence integrity.
3Measurement precision
If standard crRNA spacer length is used for Cas13 binding, then target recognition is achieved, but steric blocking efficiency is reduced
Solution Approach 1:
The invention optimizes the crRNA spacer length parameter, reducing it from the standard ~30 nucleotides to 18-24 nucleotides. This parameter change enhances the steric blocking efficiency of dCas13 while maintaining sufficient target recognition accuracy through the shortened spacer.
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
Achieves targeted modulation of regulatory elements with minimal off-target effects, effectively reversing deregulated splicing in diseases like DM1 and demonstrating improved efficacy over small RNAs and RNA interference.
Implementation Method 1
a catalytically inactive Cas13 (dCas13) protein, when recruited to a regulatory element in a nucleic acid by a CRISPR RNA (crRNA), can act as a steric blocker at the target site, thereby modulating the function of the regulatory element
Data Source
AI summary
The invention relates to modulating the function of a regulatory element in a nucleic acid, comprising delivering to a cell a catalytically inactive Cas13 (dCas13) protein and a CRISPR RNA (crRNA), wherein the crRNA recruits the dCas13 protein to the regulatory element, such that the dCas13 protein sterically blocks the regulatory element.