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Construction of MEIS2 Knockout HEK293T Cell Line by CRISPR/Cas9 Technology
Lu Lisha1, Bai Yang2, Liu Xin2, Wang Hongtao2, Gao Jie2, Yang Yiqing2,3, Su Pei2, Liu Cuicui2, Wang Yu2, Zhang Leisheng2, Xiong Tao1*, Zhou Jiaxi2*
1College of Life Sciences, Yangtze University, Jingzhou 434025, China; 2State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China; 3Hebei North University, Faculty of Medical Laboratory Sciences, Zhangjiakou 075000, China
Abstract: CRISPR/Cas9 system is the newest gene-editing technology through constitutive expression of nucleases Cas9， which binds to specific site in the genome mediated by single-guide RNA and induces doublestrand breaks (DSBs) at desired genomic loci. DSBs induced by these site-specific nucleases can be repaired by error-prone nonhomologous end joining (NHEJ) or homology directed repair (HDR)， to generate gene-specific knockout or knockin cells. MEIS2 belongs to MEIS transcription factor family with conservative homeodomains. Studies have suggested that MEIS2 is a potent regulator of development and tumorigenesis， but the mechanisms are still unclear. In this study， we designed two single-guide RNAs targeting the Exon3 and Exon8 of MEIS2 gene， which encode its two homeodomains. We measured their gene-targeting efficiency by SURVEYOR assay and Western blot analysis. Through dilution plating， we isolated a monoclonal MEIS2 knockout cell line. Sequence analysis indicated that it contained freamshift mutations in both MEIS2 alleles. In summary， we have successfully employed CRISPR/Cas9 system to construct a MEIS2 knockout cell line， and it will be a powerful tool for studying the functions and related mechanisms of MEIS2.
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