Construction and Functional Verification of human Calu3 Cell Model with Telomerase TERC Gene knockout
ZHANG Xiaona1, GUO Hongbin2, CHENG Long3, XU Shanrong4, JIN Rui3 *, TIAN Shen1 *
This study aimed to construct a human Calu3 cell model with telomerase TERC gene knockout based on an inducible DD-Cas9 system, and its function was verified. The sgRNA targeting TERC sequence was cloned into the vector pLenti-DD-Cas9-Flag, and the lentivirus was packaged and infected with Calu3 cells. The monoclonal cells obtained by screening were added with the small molecule compound TMP (trimethoprim) to induce the expression of Cas9 protein to achieve the knockout of telomerase RNA components. The telomerase activity, changes in telomere length and experiments for cell senescence staining were identified after culture. The results showed that the pLenti-DD-Cas9-Flag-sgTERC recombinant plasmid was successfully constructed, and the inducible editing of the TERC gene was completed by utilizing the high efficiency and convenience of DD-Cas9. Sequencing results showed that the TERC sequence in the monoclonal cells was partially knocked out; TRAP experiment showed that the telomerase activity was down-regulated; the cell genome was extracted and the telomere length was detected by qPCR, and the results showed that the telomere length was shortened significantly. Compared with wild-type cells, TERC knockout cells showed obvious senescence. In conclusion, deletion of TERC gene leads to the phenomenon of decreased telomerase activity, shortened telomere length and senescence in cells. Human Calu3 cell model with deletion of TERC gene can be induced, which lays a foundation for subsequent studies on cell senescence.
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