CRISPR/Cas9 LIE1 Gene Knockout for Tomato Lycopene Biosynthesis
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Solution Overview
Problem
Current methods are ineffective in significantly increasing the lycopene content in tomato fruits, which is crucial for its antioxidant and health benefits.
Innovation Solution
The CRISPR/Cas9 gene-editing technology is used to knock out the Lycopene Increasing Effectively 1 (LIE1) gene in tomato plants by introducing frameshift mutations, specifically through the insertion of a base C in the first exon and deletion of a base T in the second exon, resulting in a loss-of-function allele, thereby increasing lycopene biosynthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional breeding methods are used to increase lycopene content, then genetic diversity is maintained, but the efficiency and magnitude of lycopene increase is insufficient
Solution Approach 1:
The patent replaces conventional mechanical breeding methods (crossing, selection) with CRISPR/Cas9 gene editing technology. Specifically, the Cas9 nuclease guided by sgRNA directly targets and disrupts the LIE1 gene at its precise genomic location, achieving lycopene increase through molecular-level precision rather than traditional mechanical breeding processes
Solution Approach 2:
The patent changes the functional state of the LIE1 gene from active to inactive through targeted disruption. By introducing frameshift mutations or premature stop codons in the LIE1 coding sequence, the gene's ability to suppress lycopene biosynthesis is altered, thereby increasing lycopene content in tomato fruits
2Quantity of substance
If CRISPR/Cas9 is used to knockout LIE1 gene, then lycopene content is significantly increased, but the gene editing precision and off-target effects become critical concerns
Solution Approach 1:
The patent uses sgRNA as an intermediary molecule that bridges the Cas9 nuclease and the target LIE1 gene sequence. The sgRNA contains a 20-nucleotide spacer sequence that is complementary to a specific region of the LIE1 gene, ensuring precise targeting. This intermediary mechanism allows Cas9 to locate and cut only at the intended genomic position with high specificity
Solution Approach 2:
The patent performs preliminary design and validation of the sgRNA targeting sequence before executing the gene editing. The sgRNA sequence is selected to target a specific region of LIE1, and its specificity is verified to ensure it will not bind to off-target sites. This preliminary action prevents off-target effects before the actual gene editing occurs
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 method results in significantly higher lycopene content and redder fruit color in transgenic tomato plants, with enhanced expression of the phytoene desaturase gene, indicating a positive regulation of lycopene biosynthesis and potential health benefits.
Implementation Method 1
the RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA
Data Source
AI summary
The present disclosure provides a method for increasing lycopene content in a tomato fruit, including knocking out the gene LIE1 of SEQ ID No 1. The disclosure also provides a method for knocking out gene LIE1 in tomato. The method of the disclosure is effective for increasing the content of lycopene in tomato fruits. Finally, the disclosure provides a transgenic tomato plant with knockout of gene LIE1.