Dual-Vector CRISPR/Cas9 Repressors for In Vivo Gene Silencing

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Solution Overview

Problem

The challenge of delivering CRISPR/Cas9-based gene repressors in vivo is hindered by the size of the S. pyogenes dCas9 and KRAB domain fusion exceeding the packaging limits of standard AAV vectors, limiting their use in therapeutic applications.

Innovation Solution

A modified RNA-guided dCas9-based repressor is developed, which is customized to target any endogenous gene by designing a new guide RNA molecule, enabling efficient packaging in AAV vectors for in vivo gene regulation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If S. pyogenes dCas9-KRAB fusion is used for gene repression, then gene silencing potency is improved, but packaging capacity of AAV vector deteriorates

Engineering Contradiction:

Improvegene silencing potencyVSAvoidpackaging capacity

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The patent divides the large dCas9-KRAB fusion protein into two separate components: a small dCas9 protein packaged in one AAV vector and a separate KRAB effector domain packaged in another AAV vector. This segmentation allows each vector to remain within packaging limits while collectively delivering the full repressor function, resolving the contradiction between maintaining gene silencing potency and fitting within vector packaging capacity.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent employs a dual-vector AAV system where each vector contains essential components of the CRISPR repression system. The first vector contains dCas9 under one promoter, and the second vector contains KRAB under a different promoter, creating a nested functional system where both vectors work together to achieve complete gene repression, effectively nesting the repression function across multiple delivery vehicles.

Inventive Principle:

Principle #7Nested doll (Nesting)

2Quantity of substance

If dCas9-KRAB fusion size is reduced for AAV packaging, then packaging capacity is improved, but gene repression efficiency deteriorates

Engineering Contradiction:

Improvepackaging capacityVSAvoidgene repression efficiency

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent merges the functions of separate dCas9 and KRAB components delivered by different AAV vectors to achieve complete gene repression. Although physically separated in different vectors, the components functionally combine at the cellular level where dCas9 and KRAB interact to form the active repressor complex, thus merging their effects to maintain full repression efficiency while satisfying packaging constraints.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent creates a universal CRISPR repression system where the separated dCas9 and KRAB components can be delivered independently yet function together universally to repress any target gene guided by appropriate gRNA. This multi-functional approach allows the system to maintain full repression capability across different target genes while adapting to AAV packaging limitations through separate component delivery.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Data Source

PatentUS12428631B2CRISPR/Cas9-based repressors for silencing gene targets in vivo and methods of use

Publication Date: 2025.09.30 DUKE UNIV

AI summary

The present disclosure provides Crispr/cas9-based repressors for silencing gene targets in vivo and methods of use.