Therapeutic agent for dystrophic epidermolysis bullosa
A bad type, bullous technology, applied in gene therapy, skin diseases, tissue culture, etc., can solve the problem that there is no effective treatment for epidermolysis bullosa
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[0135] 1. Design of Genome Editing
[0136] Three types of sgRNA were prepared to select a site with good cleavage efficiency by the CRISPR-Cas9 system in the AAVS1 (adeno-associated virus integration site 1) region in the human genome. The AAVS1 region is a safe region (safe harbor) that is less susceptible to gene transfer. Since the CRISPR-Cas9 system recognizes the base sequence of "NGG" and cleaves 3 bases upstream of the sequence, regions each having "GG" at the end are selected, and each is designed to contain 20 bases upstream of "NGG". base sequence of sgRNA (sgAAVS1-#1 to #3) ( figure 1 , top; Table 1).
[0137] Table 1
[0138] sgRNA target sequence SEQ ID NO. sgAAVS1-#1 ACCCCACAGTGGGGCCACTA 3 sgAAVS1-#2 GTCACCAATCCTGTCCCTAG 4 sgAAVS1-#3 GGGGCCACTAGGGACAGGAT 5
[0139] An oligonucleotide consisting of the sequence of any one of SEQ ID NO: 3 to 5 was annealed to its complementary strand and cloned into the Bbs1 site of eS...
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