Dual-gRNA site amh gene knockout method in pelteobagrus fulvidraco and application
A yellow catfish and gene technology, applied in the fields of biotechnology and genetic breeding, can solve the problems of high cost, nonsense mutation, and high uncertainty of DNA self-repair, and achieve the effect of reducing identification cost and improving knockout efficiency
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[0036] Step (1) Target site design:
[0037] The genomic DNA sequence and its mRNA sequence of the amh gene of the yellow catfish were queried on NCBI. The full length of the amh gene of the yellow catfish was 4660bp, including 7 exons and 6 introns. Design a target site for each of the first exon and the fourth exon, the sequence of the target site is shown in Table 1, and the structure of the target site is shown in figure 1 shown. The target sequence was compared by Blast on the NCBI website to verify the specificity of the target site.
[0038] Target site selection principles:
[0039] A. The target site contains 20 bases, of which the 5' end should be GG. This is because the gRNA used in the present invention is transcribed in vitro using a T7 promoter, and the T7 promoter requires the first two digits of the transcription start site to be GG , and the third place is preferably G or A.
[0040] B. The 3 bases immediately adjacent to the 3' end of the target site cons...
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