ADAR2-CasRx Fusion Platform for Programmable RNA Editing
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Solution Overview
Problem
Existing RNA editing technologies face challenges in achieving high specificity and efficiency, particularly in mammalian cells, due to issues such as dependence on endogenous ADAR levels, immune response activation, and difficulty in targeting highly structured RNA species, with current methods often trading off between on-target and off-target editing efficiency.
Innovation Solution
The development of a novel RNA editing platform using mutated deaminase domain (dd) of RNA Adenosine Deaminase 2 (ADAR2) combined with a deactivated CRISPR-associated protein, CasRx, where specific mutations and guide RNA optimizations reduce off-target activity while maintaining high on-target efficiency, and the use of split ADAR deaminase domains further enhances specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If endogenous ADARs are recruited to target site for RNA editing, then RNA editing can be achieved, but performance cannot be controlled as it is dependent on expression level of endogenous ADARs which can be highly context-dependent
Solution Approach 1:
The patent introduces a programmable guide RNA as an intermediary that recruits ADAR enzymes to specific target sites. The guide RNA contains a spacer sequence complementary to the target RNA, enabling specific recruitment without relying on endogenous ADAR expression levels. This mediator approach decouples editing performance from cellular context
Solution Approach 2:
The patent modifies the ADAR enzyme by fusing it to a programmable domain (such as Cas13 or lambda N peptide) and altering its recruitment parameters through guide RNA design. This allows control over editing efficiency and specificity by changing guide RNA sequence and structure rather than relying on natural ADAR expression variability
2Reliability
If long or heavily chemically modified antisense oligonucleotides are used to recruit ADARs, then target site recruitment is achieved, but device complexity and ease of operation are reduced
Solution Approach 1:
The patent segments the recruitment system into modular components: a programmable RNA-binding domain (e.g., Cas13 or lambda N peptide), a guide RNA with spacer sequence, and the ADAR catalytic domain. This segmentation allows simple guide RNA design without extensive chemical modifications while maintaining reliable target recruitment
Solution Approach 2:
The patent uses a programmable RNA-binding protein or peptide as an intermediary that simplifies target recruitment. Instead of using complex chemically modified oligonucleotides, the system employs a guide RNA that directs the intermediary to the target site, reducing overall system complexity
3Adaptability or versatility
If ADAR is fused to lambda N peptide, SNAP tag, RNA-binding protein, or inactive CRISPR-associated nuclease for recruitment, then programmable target recruitment is achieved, but protein structure and stability may be affected
Solution Approach 1:
The patent employs universal programmable domains like lambda N peptide or Cas13 that can be paired with different guide RNA sequences to target multiple sites. These universal components maintain their structural stability while providing adaptable recruitment through guide RNA variation rather than protein modification
Solution Approach 2:
The patent extracts only the essential RNA-binding and recruitment functions into separate programmable domains, leaving the ADAR catalytic domain intact. This separation preserves ADAR stability while enabling programmable recruitment through the extracted functional modules
4Productivity
If existing RNA editing methods are used, then some level of editing efficiency is achieved, but off-target editing occurs and specificity is reduced
Solution Approach 1:
The patent replaces non-specific enzymatic activity with a programmable targeting system. The guide RNA provides sequence-specific recognition that directs ADAR activity only to the intended target site, substituting random enzymatic action with precision-guided catalysis
Solution Approach 2:
The patent introduces a guide RNA intermediary that mediates between the ADAR enzyme and target RNA. This intermediary ensures that editing occurs only at the intended site by providing sequence-specific binding, thereby eliminating off-target effects while maintaining high editing efficiency
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 platform achieves precise and efficient RNA editing with reduced off-target activity, capable of targeting a wide range of RNA sequences, including those previously difficult to edit, by optimizing the ADAR2-CasRx fusion and guide RNA design, leading to improved editing performance across various genetic contexts.
Implementation Method 1
ADAR (adenosine deaminase acting on RNA type 2) enzymes convert adenosine (A) to inosine (I)
Implementation Method 2
a deactivated CRISPR-associated protein, CasRx, where specific mutations and guide RNA optimizations reduce off-target activity
Data Source
AI summary
The present invention relates to artificially designed polypeptides having RNA-targeting and editing activity, wherein said polypeptides are fusion proteins comprising a modified ADAR2 deaminase domain and a Cas family targeting moiety selected from deactivated Cas13b and CasRx. Further encompassed are methods for use and uses of these polypeptides, compositions comprising them and nucleic acids encoding them as well as methods for the manufacture of said polypeptides.