Abstract
Herbicide resistance is one of the main crop traits that improve farming methods and crop productivity. CRISPR-Cas9 can be applied to the development of herbicide-resistant crops based on a target site resistance mechanism, by editing genes encoding herbicide binding proteins. The sgRNAs capable of editing the target genes of herbicides, pds (phytoene desaturase), ALS (acetolactate synthase), and EPSPS (5-Enolpyruvylshikimate-3-phosphate synthase), were designed to use with the CRISPR-Cas9 system in tomato (Solanum lycopersicum cv. Micro-Tom). The efficiency of the sgRNAs was tested using Agrobacterium mediated transient expression in the tomato cotyledons. One sgRNA designed for editing the target site of PDS had no significant editing efficiency. However, three different sgRNAs designed for editing the target site of ALS had significant efficiency, and one of them, ALS2-P sgRNA, showed over 0.8% average efficiency in the cotyledon genome. The maximum efficiency of ALS2-P sgRNA was around 1.3%. An sgRNA for editing the target site of EPSPS had around 0.4% editing efficiency on average. The sgRNA efficiency testing provided confidence that editing of the target sites could be achieved in the transformation process. We confirmed that 19 independent transgenic tomatoes were successfully edited by ALS2_P or ALS1_W sgRNAs and two of them had three base deletion mutations, which are expected to have altered herbicide resistance. In this study, we demonstrated the usefulness of performing an sgRNA efficiency test before crop transformation, and confirmed that the CRISPR-Cas9 system is a valuable tool for breeding herbicide-resistant crops.
Highlights
Weed outbreaks are the biggest problem facing agriculture worldwide [1, 2]
When glyphosate is applied to plants, it affects the primary point of action of 5-Enolpyruvylshikimate3-phosphate synthase (EPSPS) in the amino acid biosynthesis system, inhibiting the biosynthesis of aromatic amino acids such as tryptophan, tyrosine, and phenylalanine [8, 9]
Design of Single guide RNA (sgRNA) The genes involved in the response to the three most commonly used herbicides, norflurazone, sulfonylurea/imidazolinone, and glyphosate, are pds1, Acetolactate synthase (ALS), and EPSPS (5-Enolpyruvylshikimate-3-phosphate synthase), respectively
Summary
Weed outbreaks are the biggest problem facing agriculture worldwide [1, 2]. Of the 200,000 plant species on the planet, about 1% are cultivated crops, and about 0.1% are considered to be weeds. The production of herbicide-resistant crops using the CRISPR Cas9 system appears to be the preferred method of weed control to date [37]. By analyzing the amino acid sequence of target proteins, gRNA can be designed so that gene editing occurs at the site where the substrate binds, and crops with low sensitivity to herbicides can be selected from the range of edited crops produced.
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