CRISPR/Cas9 QPT2 Gene Editing for Tobacco Nicotine Reduction
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Solution Overview
Problem
Existing methods for suppressing nicotine biosynthesis in tobacco plants using GMO technology are limited in their ability to completely inhibit nicotine content, as they do not allow for 100% gene function suppression, leading to the development of 'non-GMO' tobacco with remarkably low nicotine content through CRISPR/Cas system-induced mutations in the QPT2 gene.
Innovation Solution
Genetically engineering plant cells using a CRISPR/Cas system to reduce the expression or activity of the quinolinic acid phosphoribosyl transferase (QPT) gene, specifically targeting QPT2 genes from Nicotiana species, to inhibit nicotine biosynthesis by introducing specific guide RNAs and Cas9 proteins that induce mutations in the QPT genes, thereby reducing nicotine production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If GMO technology (RNAi) is used to suppress nicotine biosynthesis, then nicotine content is reduced, but gene function suppression cannot reach 100% and nicotine content cannot be lowered sufficiently
Solution Approach 1:
The invention changes the mechanism of gene suppression from RNA interference (partial suppression) to CRISPR/Cas9-induced mutation (complete suppression). By altering the fundamental approach from reducing gene expression to creating permanent gene mutations, the patent achieves 100% suppression efficiency and reduces nicotine content to undetectable levels.
2Quantity of substance
If CRISPR/Cas system is used to induce mutation in QPT2 gene, then nicotine biosynthesis is reduced by 97% or more, but the process requires introduction of external genes and complex genetic engineering
Solution Approach 1:
The invention extracts and utilizes the plant's own DNA repair mechanisms (NHEJ and HDR pathways) to achieve gene editing. Instead of relying on complex external gene expression systems, the patent leverages endogenous cellular processes to incorporate guide RNA and Cas9 protein, thereby simplifying the overall genetic engineering approach while maintaining high efficiency.
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 effectively reduces nicotine biosynthesis by 97% or more and concurrently decreases other alkaloids like nornicotine, anabasine, and anatabine by 68% or more, resulting in plants with significantly lower carcinogenic TSNAs, such as NNK, NNN, NAB, and NAT.
Implementation Method 1
inducing a mutation in the QPT2 gene, a gene involved in nicotine biosynthesis in tobacco plants, by using a CRISPR/Cas system
Implementation Method 2
introducing specific guide RNAs and Cas9 proteins that induce mutations in the QPT genes
Implementation Method 3
effectively reduces nicotine biosynthesis by 97% or more and concurrently decreases other alkaloids like nornicotine, anabasine, and anatabine by 68% or more
Data Source
AI summary
An aspect provides plant cells with an engineered QPT gene and methods of using the same. When a QPT gene in a plant cell is genetically engineered according to an aspect, biosynthesis of nicotine is effectively inhibited, and nicotine is reduced by about 97% or more, and since alkaloids other than nicotine (nornicotine, anabasine, anatabine) are also reduced by about 68% or more, plant cells with reduced carcinogen TSNAs (NNK, NNN, NAB, and NAT) may be produced.