Alfalfa crispr/cas9 genome editing system and its application

Abstract

The invention discloses the application of the CRISPR / Cas9 genome editing system in alfalfa gene editing. The CRISPR / Cas9 genome editing system includes an expression vector, and the expression vector may include a Cas9 expression cassette, an sgRNA1 expression cassette, and an sgRNA2 expression cassette. The Cas9 expression cassette expresses Cas9. The sgRNA1 expression cassette expresses the sgRNA named sgRNA1, and the sgRNA2 expression cassette expresses the sgRNA named sgRNA2; the sgRNA1 expression cassette contains a promoter named MtU6-6promoter and the sgRNA1 gene driven by the MtU6-6promoter, The sgRNA2 expression cassette contains a promoter named MtU6‑5promoter and the sgRNA2 gene driven by the MtU6‑5promoter; the sgRNA1 and the sgRNA2 can be aimed at the same target gene or different target genes of Medicago sativa. According to the characteristics of alfalfa autotetraploid, the present invention designs dual targets for target genes, constructs a gene editing binary vector p6401-Target, and transforms alfalfa through Agrobacterium-mediated transformation to obtain regenerated plants. This optimized genome editing system has greatly improved the gene editing efficiency of alfalfa. The gene editing efficiency of plants can be as high as 100%, and the editing efficiency of a single target can be as high as 96.9%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an alfalfa CRISPR / Cas9 genome editing system and its application. Background technique [0002] Alfalfa (Medicago sativa) is an important leguminous forage crop, which has rich nutritional value, especially high protein content, high-quality dietary fiber and multivitamins, and is known as the "king of grass". Alfalfa is widely distributed in my country. In addition to its excellent feeding value, alfalfa also plays an important role in the protection and maintenance of grassland ecology. Therefore, the physiological and ecological research and genetic breeding of alfalfa have important practical significance. However, alfalfa cultivars are mostly autotetraploids with huge genomes; they are outcrossed plants with semi-self-incompatibility physiological characteristics; their genomes have high heterozygosity and difficult genetic manipulation, which poses a challenge for basic resear...

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Problems solved by technology

Another literature reported that using a tRNA structure to tandem with four gRNAs for multi-target editing of a single target gene in alfalfa, the editing efficiency can reach up to 75%. sgRNA expression, the working efficiency of a single target is still low, only 4 sgRNAs in series can realize the full editing of 4 copies of a single gene in alfalfa

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