CRISPR/Cas9 RNA Editing via Nuclear Delivery
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current RNA targeting and editing technologies, such as CRISPR/Cas systems, face challenges with efficiency, specificity, and off-target editing events, and existing methods rely on non-encodable components or require extensive protein engineering for each target, making them inefficient and costly.
Innovation Solution
Development of fully encodable and highly specific CRISPR/Cas systems comprising a nuclease-dead CRISPR-associated endonuclease (dCas) fused to a catalytically active deaminase domain of Adenosine Deaminase acting on RNA (ADAR) and an extended single guide RNA (esgRNA) for efficient and reversible RNA editing, using a viral vector like adeno-associated viral vector (AAV) for delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If antisense oligonucleotides (ASO) are used to target and manipulate RNA, then RNA manipulation capability is achieved, but the system cannot be encoded within DNA requiring regular administration throughout patient lifetime
Solution Approach 1:
The patent creates an encodable copy of the RNA targeting system by fusing the ADAR deaminase domain to a guide RNA-binding protein (such as Cas13 or CRISPR-Cas9), allowing the system to be encoded in DNA and expressed within cells, eliminating the need for repeated administration of synthetic ASOs
Solution Approach 2:
The patent extracts the catalytic function from synthetic ASOs and transfers it to an endogenous enzyme system (ADAR) that can be encoded and expressed within the cell, separating the targeting function (guide RNA) from the catalytic function (ADAR deaminase domain)
2Measurement precision
If engineered RNA binding proteins (RBP) are used to recognize target transcripts, then specific recognition and manipulation is achieved, but extensive protein engineering is required for each target making the system difficult and costly
Solution Approach 1:
The patent creates a universal platform where a single ADAR deaminase domain can be paired with different guide RNAs or guide proteins to target multiple different RNA sequences, eliminating the need to engineer new proteins for each target while maintaining high specificity through programmable guide sequences
Solution Approach 2:
The patent introduces guide RNA or guide protein as an intermediary that provides the programmable targeting function, allowing the ADAR deaminase domain to be directed to specific RNA targets without requiring engineering of the catalytic domain itself for each new target
3Adaptability or versatility
If CRISPR/Cas systems are used for RNA targeting, then programmability is achieved, but efficiency and specificity issues along with off-target editing events occur
Solution Approach 1:
The patent modifies the CRISPR/Cas system by fusing the catalytic domain to create a localized editing function at the target site, where the ADAR deaminase domain is positioned precisely at the RNA target through guide RNA complementarity, enabling site-specific editing with reduced off-target effects compared to systems that rely on broader binding interactions
Solution Approach 2:
The patent changes the catalytic parameter from nuclease activity (DNA/RNA cleavage) to deaminase activity (base modification), transforming the outcome from disruptive cleavage to precise chemical modification that maintains RNA integrity while achieving editing, thereby improving both efficiency and specificity
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 solution enables efficient, specific, and reversible RNA editing with improved targeting and reduced off-target effects, allowing for precise modulation of RNA sequences without the need for extensive protein engineering, enhancing the reliability and versatility of RNA editing processes.
Implementation Method 1
a catalytically active deaminase domain of Adenosine Deaminase acting on RNA (ADAR)... capable of CRISPR/Cas RNA-RNA base-specific Adenosine to Inosine (A-I) editing
Data Source
AI summary
Disclosed herein is a technology to perform programmable RNA editing at single-nucleotide resolution using RNA-targeting CRISPR/Cas9. This approach, which Applicants have termed “Cas9-directed RNA editing” or “CREDIT,” provides a means to reversibly alter genetic information in a temporal manner, unlike traditional CRISPR/Cas9 driven genomic engineering which relies on permanently altering DNA sequence.