A method of using small molecular compounds to improve the efficiency of genome-directed insertion
A small molecule compound, genome-targeted technology, applied in the field of genetic engineering, can solve the problems of low efficiency of HDR-mediated site-directed insertion
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Embodiment 1
[0025] Example 1: Screening of Small Molecular Compounds for Improving Genomic Site-Directed Insertion Efficiency ( figure 1 )
[0026] 1PX330-ACTB plasmid construction:
[0027] According to the gene sequence of ACTB (ENSG00000075624) provided by NCBI, a gRNA (https: / / crispr.cos.uni-heidelberg.de / index.html) was designed near its sixth exon, which is the stop codon. The primer sequences are as follows Shown: forward (F) SEQ ID No: 1: 5'-CACCCGTCCACCGCAAATGCTTCT-3'; reverse (R) SEQ ID No: 2: 5'-AAACAGAAGCATTTGCGGTGGACG-3'. After the primers were synthesized, the concentration was diluted to 10 μM, and 5 μl was mixed gently for annealing reaction. The reaction process was as follows: 95°C, 3min→10°C, 1min→95°C, 3min→10°C, 1min→95°C, 3min→10°C , 1min→95℃, 3min→4℃, 10min.
[0028] Plasmid PX330 (Addgene, #42230) was single-digested according to the instructions of ThermoFisher Company FastDigest BpiI (#FD1014), and the enzyme digestion reaction system is shown in Table 1:
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Embodiment 2
[0052] Example 2: Using Small Molecular Compounds to Improve the Efficiency of Genomic Site-directed Insertion
[0053] For the specific steps of plasmid construction and cell culture in the example, refer to Steps 1-3 of Example 1.
[0054] 4 Verification that small molecule compounds improve the efficiency of genome-directed insertion
[0055] At the same time, the optimal concentration of two candidate small molecule compounds for site-directed insertion efficiency in HEK293T cell lines was further verified, and two non-cell cycle / DNA damage compounds: Albendazole and Tedizolid. When the confluence of the cells in step 3 reaches 80-90%, co-transfect the PX330-ACTB and hACTB-T2A-GFP plasmids into HEK293T cells using Lipofectamine 3000 Reagent (#L3000015) from Shanghai Yingweijieji, the process is as follows : Prepare two 1.5mL centrifuge tubes, add 500μl Opti-MEM, 43.4μl Lipofectamine 3000 to one tube and mix well, add 14μg each of the two plasmids, 500μl Opti-MEM, 56μl P30...
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