Alfalfa CRISPR/Cas9 genome editing system and application thereof
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
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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;
[0092] MsGA3ox1-Target1-F0:
[0093] 5′-G ACACCATCCGGGGAACGAA GTTTTAGAGCTAGAAATAGC-3′, w...
Embodiment 2
[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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