Compact Cas13d RNA Targeting via Extraction and Parameter Changes
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Solution Overview
Problem
Current RNA engineering tools face limitations due to off-target effects and the difficulty in retargeting RNA-binding domains for precise RNA manipulation, particularly in human cells, where existing CRISPR-Cas systems are large and challenging to deliver effectively.
Innovation Solution
The development of a CRISPR-Cas system utilizing novel, compact Cas13d proteins and guide RNAs for efficient RNA targeting, enabling precise knockdown of endogenous RNAs and manipulation of alternative splicing, facilitated by bioinformatic analysis of prokaryotic genomes to identify suitable sequence signatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If existing CRISPR-Cas systems (Type II and VI) are used for RNA targeting, then RNA recognition capability is improved, but protein size increases to ~1200 amino acids making delivery difficult
Solution Approach 1:
The patent extracts and utilizes only the essential RNA-binding and cleavage domains from larger CRISPR-Cas systems, creating a minimized Cas13d protein that retains RNA targeting functionality while reducing size to approximately 700-900 amino acids, suitable for AAV delivery
Solution Approach 2:
The patent changes the structural parameters of the Cas13d protein by identifying and expressing truncated variants with deleted N-terminal and C-terminal regions, demonstrating that smaller protein sizes can maintain or even improve RNA binding affinity and cleavage activity
2Productivity
If RNA interference technologies are used for transcript manipulation, then RNA cleavage capability is improved, but off-target effects increase and engineering complexity rises
Solution Approach 1:
The patent replaces the RNAi mechanism (Dicer-dependent, requiring 21-23 nt siRNAs with specific structural features) with a CRISPR-Cas13d system that uses guide RNAs of 20-30 nt without requiring specific hairpin structures, enabling more precise and programmable RNA targeting with reduced off-target effects
Solution Approach 2:
The patent changes the guide RNA parameter specifications, using 20-30 nt guide RNAs without required hairpin structures, unlike RNAi which requires 21-23 nt siRNAs with specific 2-nt 3' overhangs and hairpin formations, simplifying the engineering process and improving specificity
3Adaptability or versatility
If MS2 RNA-binding domain is used for RNA targeting, then RNA binding capability is improved, but genomic modification is required to tag desired transcripts
Solution Approach 1:
The patent uses an endogenous RNA motif (U-rich sequence) as an intermediary recognition element that naturally exists in many transcripts, allowing Cas13d to target RNAs without requiring exogenous tagging systems like MS2 or Pumilio, thereby avoiding genomic modification requirements
Solution Approach 2:
The patent creates a universal RNA targeting system where Cas13d can recognize various RNA sequences through guide RNA programming without needing transcript-specific tagging, making the system broadly applicable to different RNA targets across the transcriptome
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
This approach allows for efficient and specific RNA targeting in human cells, reducing off-target effects and enabling precise RNA manipulation, paving the way for advanced transcriptome engineering applications.
Implementation Method 1
at least one CRISPR-Cas system guide nucleic acid molecule (such as a guide RNA, gRNA) that hybridizes with the one or more target RNA molecules
Implementation Method 2
the gRNA directs the complex to the one or more target RNA molecules and modifies (e.g., cuts, detects) the one or more target RNA molecules
Data Source
AI summary
Provided herein are CRISPR/Cas methods and compositions for targeting RNA molecules, which can be used to detect, edit, or modify a target RNA.