Chemically Modified CRISPR-Cas13 crRNA for Sustained RNA Knockdown
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
CRISPR-Cas13-mediated RNA knockdown effects in human cells are short-lived due to rapid crRNA degradation by endogenous RNA nucleases, making it challenging to achieve lasting RNA manipulation without genetic modification, which is undesirable for therapeutic settings.
Innovation Solution
Chemically modified crRNAs with modifications such as 2′-O-methyl, 3′phosphorothioate, and 3′ inverted thymine are used to enhance the stability and specificity of crRNA, allowing for prolonged RNA targeting and knockdown efficacy when combined with Cas13 polypeptides or recombinant expression vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of moving object
If unmodified crRNA is used for Cas13-mediated RNA knockdown, then the system is simple and easy to deliver, but the crRNA is rapidly degraded by endogenous RNA nucleases resulting in short-lived effects
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of crRNA nucleotides through various chemical modifications (2′-O-methyl, 3′phosphorothioate, 2′O-methyl-3′-phosphorothioate, and 3′ inverted thymine). These chemical parameter changes to the crRNA molecule enhance its resistance to endogenous RNA nucleases, thereby extending the duration of RNA knockdown effects from transient to sustained periods without requiring continuous crRNA expression
Solution Approach 2:
The patent employs composite materials by creating chemically modified crRNA molecules that combine different modified nucleotide types in specific configurations. The use of composite chemical modifications (combining 2′-O-methyl with 3′phosphorothioate, for example) creates crRNA structures with enhanced stability properties that surpass unmodified crRNA, achieving prolonged RNA manipulation effects
2Reliability
If chemically modified crRNA is used to extend transient Cas13-mediated effects, then the stability and duration of RNA knockdown is improved, but the complexity of crRNA synthesis and delivery increases
Solution Approach 1:
The patent utilizes parameter changes by systematically varying the chemical modification parameters of crRNA nucleotides. Different modification types (2′-O-methyl, 3′phosphorothioate, etc.) and their positions along the crRNA sequence are optimized to achieve the desired balance between stability and manufacturability. This parameter optimization allows for reliable crRNA synthesis through established chemical modification protocols
3Duration of action of stationary object
If continuous crRNA expression is achieved via genetic manipulation, then lasting RNA manipulation is possible, but genetic modification of host DNA is required which is undesirable for therapeutic settings
Solution Approach 1:
The patent extracts the requirement for genetic manipulation by using chemically modified crRNA molecules that provide sustained stability without needing to be genetically encoded. The chemically modified crRNA can be delivered as a discrete molecule that persists long enough to achieve lasting RNA manipulation effects, thereby separating the duration extension function from genetic modification
Solution Approach 2:
The chemically modified crRNA acts as an intermediary that bridges the gap between transient unmodified crRNA and permanent genetic manipulation. These modified molecules provide intermediate-duration stability, enabling sustained RNA manipulation effects without requiring integration into the host genome, thus avoiding the harmful effects of genetic modification while achieving lasting therapeutic effects
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 modified crRNAs significantly improve RNA targeting efficiency and stability, enabling sustained gene expression modulation in human cells and primary immune cells, including effective knockdown of SARS-CoV-2 virus, thus providing a robust and efficient method for RNA manipulation without genetic modification.
Implementation Method 1
The crRNA comprises one or more chemically modified nucleotides. In one embodiment, the crRNA comprises 1, 2, 3, 4, or 5 modified nucleotides at the 3′ end of the spacer sequence.
Implementation Method 2
CRISPR-based transcriptome modulation has been proposed to offer considerable therapeutic potential for a wide spectrum of RNA-mediated diseases. In all cases, the Cas13-mediated effects are directed by CRISPR RNAs (crRNA) that guide Cas13 to the target RNA by RNA-RNA hybridization of a short spacer sequence ( ̃20-30 nt) to the target site.
Data Source
AI summary
Provided herein are compositions and methods comprising modified crRNA comprising a spacer sequence and a direct repeat sequence, wherein the crRNA comprises one or more chemically modified nucleotides. Also provided are methods of enhancing modulation of gene transcripts in a cell, the method comprising introducing into the cell a modified crRNA as described herein, and a Cas13 polypeptide, an mRNA encoding a Cas13 polypeptide, and/or a recombinant expression vector comprising a nucleotide sequence encoding a Cas13 polypeptide, wherein the modified crRNA guides the Cas13 polypeptide to the target RNA sequence, and wherein the modified crRNA induces regulation of the target RNA with an enhanced activity relative to a corresponding unmodified crRNA.