Short Multi-Repeat RNA Constructs for Precise Gene Silencing
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Solution Overview
Problem
Current CRISPR technologies for DNA editing face limitations in targeting and editing multiple genes, leading to unpredictable phenotypes and off-target effects, and require the introduction of 'foreign' bacterial nucleic acids, which is not ideal for agricultural applications.
Innovation Solution
A programmable RNA-guided targeting system using short multiple repeat single-stranded antisense sequences that are complementary to target RNA, avoiding the use of CRISPR nuclease proteins and traditional transgenic technology, leveraging endogenous RNAi machinery for precise gene silencing in plants and other organisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR nuclease proteins and traditional transgenic technology are used for gene editing, then DNA editing capability is achieved, but foreign bacterial nucleic acids are introduced and off-target effects occur
Solution Approach 1:
The invention extracts and utilizes only the guide RNA component from the CRISPR system, eliminating the need for CRISPR nuclease proteins. This allows RNA-targeting capability to be retained while removing the harmful introduction of foreign bacterial nucleic acids and reducing off-target effects associated with traditional CRISPR DNA editing.
Solution Approach 2:
The invention uses short multiple repeat single-stranded antisense RNA sequences as intermediaries that bridge the guide sequence and target RNA. These repeat sequences facilitate precise RNA-RNA hybridization and recruitment of endogenous RNAi machinery, achieving specific gene silencing without requiring foreign CRISPR nucleases.
2Adaptability or versatility
If CRISPR systems are used for multigene trait editing, then multiple genes can be targeted, but unpredictable pleiotropic phenotypes occur
Solution Approach 1:
The invention segments the gene silencing function into multiple independent short repeat RNA sequences, each targeting a specific gene. This allows precise control over which genes are silenced and at what levels, enabling multigene trait editing with predictable phenotypic outcomes by independently tuning each guide RNA's expression.
Solution Approach 2:
The invention changes the parameter of target specificity from DNA to RNA level, allowing temporal and spatial control over gene silencing. By targeting RNA rather than editing DNA permanently, the system enables reversible and conditionally controllable multigene silencing, improving phenotype predictability through parameter adjustment.
3Reliability
If existing RNA interference technologies are used for transcript inhibition, then gene silencing is achieved, but significant off-target effects occur
Solution Approach 1:
The invention applies local quality by designing short repeat sequences with specific local complementarity to the target RNA. Each repeat sequence is optimized to match a specific region of the target transcript, creating localized and precise silencing effects that minimize off-target binding and improve specificity compared to traditional RNAi approaches.
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 achieves high levels of target RNA silencing with reduced off-target effects, allowing for precise manipulation of endogenous and viral RNA, enabling improved agricultural productivity and resistance to pathogens without introducing foreign DNA.
Implementation Method 1
two or more distinct guide nucleotide sequences that are complementary to one or more RNA targets
Implementation Method 2
leveraging endogenous RNAi machinery for precise gene silencing
Data Source
AI summary
Short multi-repeat RNA targeting constructs for manipulating RNA targets and obtaining variable levels of gene silencing, and methods of using the same to silence RNA targets in mammals, insects, plants, and fungus. The constructs comprise two or more distinct guide nucleotide sequences (in the absence of CRISPR nuclease) that are complementary to one or more RNA targets, wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 100 nt or less.