Cas9 fused protein capable of improving gene knock in efficiency and exogenous gene knock in and integration system
A fusion protein, gene knock-in technology, applied in the field of genetic engineering, can solve the problem of unfavorable cell growth due to site/cell difference, and achieve the effect of improving knock-in efficiency, facilitating gene therapy, and promoting KI events
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Embodiment 1
[0044] 1. Construction of PX330-gRNA-Cas9-DBD vector
[0045] (1) Design and synthesize primers for PCR amplification according to the gene CDS sequence of the transcription factor DNA binding domain provided by NCBI (primers are shown in Table 1). For the PCR system and reaction procedures, refer to Takara Company’s Max DNAPolymerase (#R045A) instruction manual, recovery and purification of PCR products. For the method of recovering and purifying the PCR product, refer to the instructions of the Gel Extraction Kit (#D2500) of Omega Company.
[0046] Table 1 DBD primer information table
[0047]
[0048]
[0049] (2) The map of the PX330-HindIII plasmid (PX330-HindIII plasmid is as follows figure 2 shown) according to the instructions of ThermoFisher FastDigest HindIII (#FD0504) for single enzyme digestion. The digested plasmid and the PCR product purified in step (1) were seamlessly cloned. For the detailed cloning process, refer to the manual of In-Fusion HD Cloni...
Embodiment 2
[0090] 1. The effect of a single drug on KI efficiency
[0091] The PX330-hACTB-Cas9-mTHAP11 vector and the 2mTHAP11-hACTB-2mTHAP11 donor vector were co-transfected into HEK293T cells (10cm cell plate). After 6 hours of transfection, the cells were digested and inoculated into a 24-well plate at a ratio of 1:1. After 12 hours, the small molecule compound was added after the cells adhered to the wall, and the final concentration was controlled to be 10 μM, and only one drug was contained in each well. After 36-48 hours, the expression of green fluorescence was detected by flow cytometry, which was the KI efficiency of the cells. Two better small molecule compounds screened out were Vinblastine and Valnemulin hydrochloride. The above-mentioned experimental process was repeated after the drug was serially diluted, and the final concentration of the drug was set in four gradients: 10 μM, 5 μM, 2.5 μM, and 1 μM. The result is as Figure 13 As shown, Vinblastine and Valnemulin hy...
Embodiment 3
[0097]Co-transfect the PX330-hACTB-Cas9-mTHAP11 vector and the donor vector 2mTHAP11-hACTB-2mTHAP11, and co-transfect the PX330-hGAPDH-Cas9-mTHAP11 vector and the donor vector 2mTHAP11-hGAPDH-2mTHAP11 into different cell lines, namely HEK293T cells, Hela cells, HepG2 cells, and A549 cells were digested 6 hours after transfection, and seeded into 24-well plates at a ratio of 1:1. After 12 hours, after the cells adhered to the wall, Valnemulin was added to various KI cells. 1 , control the final concentration to 1 μM, and use flow cytometry to detect the expression of green fluorescence after 36-48 hours to verify that the Cas9-mTHAP11 fusion protein cooperates with Valnemulin 1 Effects on KI efficiency for different genomes and different cell lines. The result is as Figure 16-19 As shown, in HEK293T cells, Hela cells and HepG2 cells, Cas9-mTHAP11 fusion protein cooperates with Valnemulin 1 Both can improve the KI efficiency of the exogenous gene knock-in integration system a...
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