Gene knockout method for breeding selection of fhl1b gene-deletion type zebra fish
A gene deletion and gene knockout technology, applied in the field of gene knockout, can solve the problems of high off-target rate and low efficiency of targeting technology
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[0078] 1) Design CRISPR / Cas9 gene knockout target sites and detection primers
[0079] On the National Center for Biotechnology Information (NCBI), query the genomic DNA sequence of the zebrafish fhl1b gene, on the website SMART ( http: / / smart.embl-heidelberg.de / ) to analyze its functional domains, according to the principle of CRISPR / Cas knockout, on the website The ZiFiT Targeter ( http: / / zifit.partners.org / ZiFiT / ) to design the target site of fhl1b gene. The selection of targets must follow this standard: 5'-GG-(N)18-NGG-3'. The GG dinucleotide at the 5' end is part of the T7 promoter, and this restriction is not required when designing the target site, but it must be ensured that the 3' end of the target site is NGG. The selected position of the target point must be within the structural domain of the gene to ensure that the insertion or deletion of bases at the target site can affect the entire structural domain of the fhl1b gene, thereby changing the expression o...
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