Aptamer-Recruited CRISPR Editors for Single-AAV Multiplexing
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Solution Overview
Problem
Existing CRISPR editors face challenges in multiplexing distinct functions at multiple genomic sites due to limited orthogonal and inducible mediators and inefficient intracellular delivery, particularly due to their large size exceeding the capacity of a single adeno-associated virus (AAV) capsid.
Innovation Solution
Development of miniaturized CRISPR editors with intracellularly evolved RNA aptamers that recruit endogenous effectors, allowing for compact and multiplexable genomic editing, packaged into a single AAV capsid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If existing non-nuclease CRISPR editors are used for multiplexed genomic editing, then distinct functions can be performed at multiple genomic sites, but the system size exceeds the capacity of a single AAV capsid, resulting in inefficient intracellular delivery
Solution Approach 1:
The patent segments the effector recruitment function by using separate aptamer-RNA complexes that can be independently delivered and assembled at the genomic site. Instead of delivering a large monolithic editor system, the invention uses discrete aptamer units that recruit effectors locally, enabling multiplexed editing while reducing the size of individual delivery components below AAV capsid capacity limits.
2Adaptability or versatility
If existing CRISPR editors are used for multiplexing, then multiple genomic sites can be targeted, but the number of orthogonal and inducible mediators is limited
Solution Approach 1:
The patent creates a universal mediator system where a single aptamer-RNA complex architecture can recruit multiple different effectors (transcriptional activators, repressors, epigenetic modifiers) through modular effector fusion. This universal aptamer platform enables multiplexed editing at multiple genomic sites without requiring separate orthogonal mediators for each function, reducing overall system complexity.
3Ease of operation
If RNAi technology is used for sequence-specific gene silencing, then gene expression can be regulated, but efficiency and specificity require further improvement and the editing is transient
Solution Approach 1:
The patent replaces the RNAi mechanism with a direct DNA-targeting CRISPR-Cas system that operates at the genomic level. This substitution provides more efficient and persistent editing by directly modifying DNA sequences rather than relying on transient RNA interference, enabling both sequence-specific silencing and long-term genomic editing through stable integration or heritable changes.
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
Enables efficient, multiplexed genomic editing at transcriptional, epigenetic, and post-translational levels, facilitating the investigation of complex gene networks and therapeutic applications such as understanding drug resistance in cancer cells.
Implementation Method 1
RNA sequences that can bind to effectors through molecular recognition
Implementation Method 2
guide sequence capable of hybridizing to a target sequence
Data Source
AI summary
In one aspect, the disclosure relates to multiplexable, non-nuclease CRISPR editors comprising RNA aptamer sequences configured to bind to endogenous effector molecules. The disclosed CRISPR editors are small in size and can be delivered by a single adeno-associated virus capsid. Also disclosed are a method for intracellular evolution of aptamers, a method for introducing a genomic modifying event to a host cell using the disclosed CRISPR editors, and a host cell modified by the disclosed methods. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present disclosure.