Partially Single-Stranded Donor Molecules for Targeted Genome Integration
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Solution Overview
Problem
Current methods for targeted genome modification in plants are inefficient and unpredictable, often resulting in random integration of exogenous DNA, making it difficult to achieve precise and specific insertion of desired traits, and are labor-intensive and costly.
Innovation Solution
The use of donor nucleic acid molecules with short single-stranded complementary regions that integrate into the genome via the non-homologous end joining (NHEJ) pathway, facilitated by zinc finger nucleases or TALE nucleases, allowing for precise insertion of exogenous sequences at specific locations without undesired sequence alterations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional transformation methods are used to deliver exogenous DNA into plant genomes, then DNA integration occurs, but the integration location is random and screening is required
Solution Approach 1:
The patent changes the molecular parameters of the donor DNA from double-stranded to partially single-stranded with 3' overhangs, which fundamentally alters the integration mechanism from random to targeted, eliminating the need for screening thousands of transformants
Solution Approach 2:
The patent introduces zinc finger nucleases or TALE nucleases as intermediary molecules that create site-specific double-strand breaks in the target genome, serving as a bridge between the donor DNA and the integration site to enable precise targeted integration
2Manufacturing precision
If donor DNA with long homology arms is used for targeted integration, then integration accuracy improves, but the complexity of donor molecule construction increases
Solution Approach 1:
The patent extracts only the essential single-stranded 3' overhang regions from the complete homology arm structure, retaining the targeting function while eliminating the need for long double-stranded homology arms, thus simplifying donor molecule construction
Solution Approach 2:
The patent employs asymmetric donor DNA structure with single-stranded 3' overhangs on one end and blunt or different overhangs on the other end, which directs the integration orientation and simplifies the design compared to symmetric long homology arms
3Reliability
If site-specific nucleases are used to create targeted DSB, then integration specificity improves, but the risk of off-target effects increases
Solution Approach 1:
The patent uses partial single-stranded overhangs rather than complete strand separation, and employs nucleases that create controlled DSBs with specific overhang patterns, reducing off-target effects while maintaining integration specificity
Solution Approach 2:
The patent converts the potentially harmful off-target nuclease activity into a benefit by designing the donor DNA with overhangs that specifically match the intended target site pattern, allowing only correctly targeted integrations to succeed while mismatched off-target events are eliminated
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 targeted and efficient integration of desired sequences into the plant genome, reducing the need for extensive screening and improving the predictability and specificity of trait introduction, thereby streamlining the process of plant trait engineering.
Implementation Method 1
facilitated by zinc finger nucleases or TALE nucleases
Implementation Method 2
facilitated by zinc finger nucleases or TALE nucleases
Implementation Method 3
integrate into the genome via the non-homologous end joining (NHEJ) pathway
Implementation Method 4
The single-stranded complementary regions anneal to the 5' overhang of the target sequence created by nuclease cleavage
Data Source
AI summary
Disclosed herein are donor molecules comprising single-stranded complementary regions flanking one or more sequences of interest. The donor molecules and/or compositions comprising these molecules can be used in methods for targeted integration of an exogenous sequence into a specified region of interest in the genome of a cell.