Modular CRISPR-Cas Cloning System for Orphan Crop Genome Editing
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Solution Overview
Problem
There is an unmet need for effective technologies to precisely edit genes and genomes in orphan crops to impart desired traits such as pest and disease resistance, nutrient biofortification, and drought or flood tolerance, as existing methods lag behind in application for these crops compared to major crops like maize, soybean, and wheat.
Innovation Solution
A gene editing cloning system utilizing CRISPR-Cas technology, comprising expression cassettes with guide RNAs, a CRISPR-associated protein, and selectable markers, is developed to efficiently transform plants, allowing for targeted mutagenesis and trait acquisition in orphan crops like cassava, cowpea, and millet.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If gene editing technologies are applied to orphan crops, then precision and efficiency of genome editing is improved, but the complexity of the cloning system increases
Solution Approach 1:
The cloning system is divided into multiple modular expression cassettes, each containing specific functional elements (promoter, gRNA scaffold, guide sequence, terminator). These standardized modules can be independently designed, assembled, and exchanged to create different gene editing configurations without redesigning the entire system, thus maintaining precision while managing complexity through systematic organization.
Solution Approach 2:
The expression cassettes are designed with universal components that can function across different orphan crop species. The standardized promoter-gRNA-scaffold-terminator structure serves multiple purposes: it enables precise target gene identification, facilitates efficient Cas9 protein recruitment, and allows multiplexed editing of multiple genes simultaneously. This multi-functional design achieves high editing precision without proportionally increasing system complexity.
2Productivity
If multiple expression cassettes with gRNAs and Cas protein are used, then the efficiency of plant transformation is improved, but the device complexity increases
Solution Approach 1:
The transformation system is segmented into independent expression cassettes that can be assembled in a standardized manner. Each cassette contains a promoter, gRNA scaffold, guide sequence, and terminator, allowing modular construction of multi-gene editing systems. This segmentation enables efficient transformation by allowing independent optimization of each cassette while maintaining overall system manageability through repetitive modular units.
Solution Approach 2:
The gRNA scaffolds and promoter-terminator sequences are pre-designed and standardized before the actual gene editing experiment. This preliminary preparation of modular components allows researchers to quickly assemble different gene editing configurations without repeatedly designing the entire system, thus improving transformation efficiency while keeping the cloning process systematic and manageable.
3Ease of operation
If standardized expression cassettes with promoters and terminators are implemented, then the ease of operation is improved, but the loss of time in cloning process may increase due to assembly steps
Solution Approach 1:
The cloning system uses segmented expression cassettes with standardized boundaries and interfaces. This segmentation allows for pre-prepared modular units to be assembled through standardized protocols, making the cloning process more straightforward and easier to operate. The modular nature enables researchers to focus on selecting and assembling pre-validated components rather than designing each element from scratch, improving ease of operation despite the assembly steps required.
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
The system enables precise editing of plant genomes to confer traits like pest resistance, nutrient biofortification, and drought tolerance, enhancing the agricultural self-sufficiency of orphan crops through efficient and targeted genome modification.
Implementation Method 1
the gRNA comprises at least 15 nucleotides guide sequence complementary to a target gene sequence
Data Source
AI summary
The present disclosure relates to compositions, systems, and kits for modifying a gene sequence of interest in plants and producing a gene-edited plant, part, or cell. The present disclosure further relates to methods for obtaining a gene sequence of interest to target using a CRISPR-Cas9 system, and methods for introducing such gene modification into plants.