CRISPR Editing of Wheat Glu-1Ax-null Gene to Restore HMW-GS
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current traditional breeding methods for increasing the number of expressed high-molecular-weight glutenin subunits (HMW-GS) in wheat are hindered by linkage drag, leading to long breeding periods, high costs, and low efficiency, which affects the processing quality of wheat.
Innovation Solution
The method involves precise editing of the Glu-1Ax-null gene in wheat using the CRISPR/SpCas9 system, specifically designing a single-stranded guide RNA (sgRNA) to target and disrupt a premature termination codon, thereby activating the expression of the silenced Glu-1Ax-null subunit and increasing the number of expressed HMW-GS.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional breeding methods are used to increase the number of expressed HMW-GS, then the number of expressed HMW-GS can be increased, but the breeding period becomes long, cost increases, and efficiency decreases
Solution Approach 1:
The patent replaces traditional mechanical breeding methods (crossing, back-crossing) with gene editing technology (CRISPR/SpCas9 system). This substitution enables precise modification of the Glu-1Ax-null gene to remove the premature termination codon, directly activating expression of the silenced HMW-GS without requiring multi-generation breeding, thereby dramatically shortening the breeding period while increasing the number of expressed HMW-GS
Solution Approach 2:
The patent changes the genetic parameter of the Glu-1Ax-null gene by editing the premature termination codon (TAA) to restore the reading frame and enable full-length protein expression. This parameter change (gene sequence modification) directly activates the silenced subunit expression, increasing the number of expressed HMW-GS from 3-5 to potentially all 6 theoretical subunits without the time-consuming breeding process
2Quantity of substance
If traditional breeding methods are used to increase the number of expressed HMW-GS, then the number of expressed HMW-GS can be increased, but the cost increases and efficiency decreases
Solution Approach 1:
The patent replaces inefficient traditional breeding mechanics with precise gene editing mechanics. The CRISPR/SpCas9 system allows direct modification of the Glu-1Ax-null gene in a single generation, eliminating the need for multiple back-crossing cycles. This substitution dramatically improves breeding efficiency by reducing the number of generations required from multiple cycles to essentially one generation, while also reducing associated costs
3Quantity of substance
If traditional breeding methods are used to increase the number of expressed HMW-GS, then the number of expressed HMW-GS can be increased, but linkage drag occurs affecting other agronomic traits
Solution Approach 1:
The patent replaces traditional breeding that relies on recombination and segregation with precise gene editing. By directly modifying only the Glu-1Ax-null gene to remove the premature termination codon, the method achieves activation of the silenced subunit without bringing along linked genes from donor parents. This eliminates linkage drag that would otherwise affect other agronomic traits, allowing independent modification of the target gene without unwanted side effects
Data Source
AI summary
The present invention provides a method for activating expression of a Glu-1Ax-null subunit in wheat by gene editing, the method comprising: designing a specific sgRNA for targeting a premature termination codon in a Glu-1Ax-null gene of wheat; cloning the specific sgRNA into a pair of BsaI sites of a vector pEtRNA to construct a CRISPR/SpCas9 editing vector for the Glu-1Ax-null gene; and transforming the CRISPR/SpCas9 editing vector for the Glu-1Ax-null gene into wheat cell via Agrobacterium-mediated transformation and selecting a homozygous line in which the premature termination codon is disrupted. The inventive specific sgRNA is designed near the 1216-1218-site premature termination codon of the Glu-1Ax-null gene, the CRISPR/SpCas9 editing vector is transformed into wheat via Agrobacterium-mediated transformation, and the premature termination codon can be specially disrupted, thereby activating expression of silenced high-molecular-weight glutenin Glu-1Ax-null subunit in wheat.