Tissue-Specific Guide RNA Design for CRISPR Off-Target Reduction
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Solution Overview
Problem
Current RNA-targeted endonuclease technologies, such as CRISPR/Cas, face challenges in achieving efficient and specific cell and tissue targeting, leading to off-target effects and limited clinical applicability due to poor biodistribution and payload capacity restrictions.
Innovation Solution
The development of novel methods to design and synthesize cell and tissue-specific guide RNAs (gRNAs) that hybridize with target nucleic acid sequences within specific cell types, allowing for precise gene editing while minimizing off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional delivery technologies such as LNPs or AAVs are used for tissue specific gene editing, then delivery capacity is improved, but off-target effects and uncontrolled biodistribution occur
Solution Approach 1:
The patent applies local quality by designing gRNAs with tissue-specific targeting capabilities, where each gRNA is customized to target specific cell types or tissues. This allows the gene editing system to exhibit different targeting properties in different locations, achieving both high payload capacity through conventional delivery vehicles and minimal off-target effects through localized specificity.
Solution Approach 2:
The gRNA serves as an intermediary between the delivery vehicle and the target genome. It mediates the interaction by recognizing specific target sequences and guiding the Cas enzyme to the correct location, thereby enabling tissue-specific editing while preventing off-target effects even when using high-capacity delivery systems like LNPs or AAVs.
2Measurement precision
If vector targeting is used to achieve tissue specificity, then targeting precision is improved, but vector design complexity and payload capacity restrictions worsen
Solution Approach 1:
The patent segments the targeting function from the delivery vehicle. Instead of designing complex tissue-specific vectors, it separates the delivery component (which can use simple LNPs or AAVs) from the targeting component (gRNA). This segmentation allows each component to be optimized independently, reducing overall design complexity while maintaining high targeting precision.
Solution Approach 2:
The gRNA system provides universal applicability across different delivery vehicles. The same gRNA can be combined with various delivery methods (LNPs, AAVs, or other vehicles) to achieve tissue-specific editing, making the system multi-functional and adaptable without requiring custom vector design for each delivery platform.
3Measurement precision
If AAVs are used for tissue targeting, then tissue specificity is improved, but payload capacity is restricted to less than 5 kb of DNA
Solution Approach 1:
The patent extracts the targeting function from the AAV payload and places it in the gRNA. This allows the AAV to deliver only the essential Cas enzyme protein (small payload) while the gRNA, delivered separately or in a different AAV, provides the tissue-specific targeting. This extraction resolves the payload capacity limitation while maintaining AAV tissue specificity.
Solution Approach 2:
The system uses a nested approach where the gRNA can be delivered within or alongside the AAV particle, with the gRNA serving as a nested component that enables targeting without increasing the AAV payload capacity. The gRNA is compact and can be packaged efficiently, allowing the AAV to maintain its size constraints while gaining enhanced targeting capability.
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 enables highly specific gene editing in intended cell types with minimal editing in unintended cells or tissues, enhancing the safety and efficacy of in vivo RNA-guided endonuclease complexes.
Implementation Method 1
the gRNA comprises a nucleic acid sequence that is configured to hybridise with a target nucleic acid sequence within the genome of a cell
Data Source
AI summary
Methods are provided for making a guide RNA (gRNA) suitable for use within a RNA-guided endonuclease complex, wherein the gRNA comprises a nucleic acid sequence that is configured to hybridise with a target nucleic acid sequence within the genome of a cell, and wherein the target nucleic acid sequence is characterised as being comprised within a locus that is active within a specific cell type. Also provided are RNA-guided endonuclease complexes and cell or tissue type selective or preferential gRNAs.