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Generation of JAK2 Gene Knockout K562 Cell Line by CRISPR/Cas9 System |
Lu JIAN,Ying-hui HUANG,Tian-ya LIANG,Li-min WANG,Hong-tao MA,Ting ZHANG,Dan-yang LI,Ming-lian WANG() |
Beijing University of Technology, Beijing 100020, China |
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Abstract Objective: To generate a JAK2 knockout K562 cell line by CRISPR/Cas9 gene editing system.Methods: Using CRISPR online design tool, sgRNA was designed targeted to JAK2 gene and used to construct the co-expression plasmid of Cas9-sgRNA. Lentivirus was packaged by the second-generation lentivirus packaging system and used to transduce K562 cells, the genomic DNA of cell pool was extracted, and the gene editing activity was detected by Sanger sequencing and TA cloning. The candidate edited cells were inoculated into 96-well plate by infinite dilution method. Then the cells were expanded to extract genomic DNA. The JAK2 sequence was identified by Sanger sequencing and TA cloning.Results: LentiCRISPRv2-sgRNA3-1 plasmid containing the gene editing tools for koncking out JAK2 was constructed. The optimal dose of lentivirus with low cytotoxicity and high transduction efficiency was obtained. The JAK2 gene knockout K562 cell line (K562-JAK2 -/-) was successfully generated.Conclusion: Lentivirus transduction-based CRISPR/Cas9 system was successfully used to generate JAK2 gene homozygous knockout K562 cell line, providing a cell model to study the importance of JAK2 gene in the field of chronic myeloid leukemia. Besides, the lentivirus transduction-based CRISPR/Cas9 protocol reported here lays a foundation for constructing other gene knockout K562 cell lines to study of hematopoietic differentiation mechanism.
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Received: 06 December 2018
Published: 05 August 2019
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Corresponding Authors:
Ming-lian WANG
E-mail: mlw@bjut.edu.cn
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