Alfalfa CRISPR/Cas9 genome editing system and application thereof
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A genome editing and alfalfa technology, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of low work efficiency of a single target
Active Publication Date: 2021-10-12
CHINA AGRI UNIV
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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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Embodiment 1
[0085] Example 1. Using the optimized CRISPR / Cas9 system to edit the MsGA3ox1 gene of alfalfa to obtain dwarf plant materials
[0086] 1. Construction of gene editing vector p6401-MsGA3ox1
[0087] 1. Gene editing target design
[0088] The coding sequence (CDS) of Medicago MsGA3ox1 is the MsGA3ox1 coding gene shown in sequence 2 in the sequence listing. According to the genome sequence of Alfalfa MsGA3ox1, through the online target prediction website ( http: / / crispor.tefor.net / ) to generate a target list, select target 1 as 5′-ACACCATCCGGGGAACGAA-3, and target 2 as 5′-AAGCCATTCGTTCCCCGGA-3′.
[0089] 2. Construction of sgRNA module
[0090] Primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.
[0091] MsGA3ox1-Target1-BsF: 5′-ATATATGGTCTCGCTTG ACACCATCCGGGGAACGAA GTT-3', wherein the 18th to 36th nucleotides are the designed MsGA3ox1 target 1 sequence;
[0125] Example 2. Using the optimized CRISPR / Cas9 system to edit the MsNP1 gene of alfalfa to obtain male sterile materials
[0126] 1. Construction of gene editing vector p6401-MsNP1
[0127] 1. Gene editing target design
[0128] The coding sequence (CDS) contained in Medicago MsNP1 is the MsNP1 coding gene shown in sequence 3 in the sequence listing. According to the alfalfa MsNP1 genome sequence, through the online target prediction website ( http: / / crispor.tefor.net / ) to generate a target list, select target 1 as 5′-GCTACATTGAAGCCGCTAG-3′, and target 2 as 5′-TCCTTTTTGAGAACCTGTGA-3′.
[0129] 2. Construction of sgRNA module: primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.
[0130] MsNP1-Target1-BsF: 5′-ATATATGGTCTCGCTTG GCTACATTGAAGCCGCTAG GTT-3', wherein the 18th to 36th nucleotides are the designed MsNP1 target 1 sequence;
[0131] MsNP1-Target1-F0:5′-G GCTACATTGAAGCCGCTAG GTTTTAGAGCTAGAAATAGC-3′, wherein the 2nd to 20th nucleotides ar...
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Abstract
The invention discloses application of a CRISPR/Cas9 genome editing system in alfalfa gene editing, the CRISPR/Cas9 genome editing system comprises an expression vector, the expression vector can comprise a Cas9 expression cassette, an sgRNA1 expression cassette and an sgRNA2 expression cassette, and the Cas9 expression cassette is used for expressing Cas9. The sgRNA1 expression cassette is used for expressing sgRNA with the name of sgRNA1, and the sgRNA2 expression cassette is used for expressing sgRNA with the name of sgRNA2; the sgRNA1 expression cassette contains a promoter with the name of MtU6-6promoter and a sgRNA1 gene driven by the MtU6-6promoter, and the sgRNA2 expression cassette contains a promoter with the name of MtU6-5promoter and a sgRNA2 gene driven by the MtU6-5promoter; the sgRNA1 and the sgRNA2 can aim at the same target gene or different target genes of the alfalfa. According to the invention, double targets are designed for a target gene according to the characteristics of an autotetraploid of alfalfa, a gene editing binary vector p6401-Target is constructed, and alfalfa is subjected to agrobacterium-mediated transformation to obtain a regenerated plant. According to the optimized genome editing system, the alfalfa gene editing efficiency is greatly improved, the plant gene editing efficiency can reach up to 100%, and the single target editing efficiency can reach up to 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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