Generation of <i>JAK2</i> Gene Knockout K562 Cell Line by CRISPR/Cas9 System

doi:10.13523/j.cb.20190706

China Biotechnology

2019, Vol. 39

Issue (7): 39-47 DOI: 10.13523/j.cb.20190706

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.

Key words： CRISPR/Cas9 system JAK2 gene K562 cells Lentivirus

Received: 06 December 2018 Published: 05 August 2019

ZTFLH:

Q789

Corresponding Authors: Ming-lian WANG E-mail: mlw@bjut.edu.cn

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	Articles by authors
	Lu JIAN
	Ying-hui HUANG
	Tian-ya LIANG
	Li-min WANG
	Hong-tao MA
	Ting ZHANG
	Dan-yang LI
	Ming-lian WANG

Cite this article:

Lu JIAN,Ying-hui HUANG,Tian-ya LIANG,Li-min WANG,Hong-tao MA,Ting ZHANG,Dan-yang LI,Ming-lian WANG. Generation of JAK2 Gene Knockout K562 Cell Line by CRISPR/Cas9 System. China Biotechnology, 2019, 39(7): 39-47.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190706 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I7/39

Fig.1 Plasmid construction and identification of lenticrisprv2-sgRNA3-1 (a) Agarose gel electrophoresis of lentiCRISPRv2 cut by BsmbI Lane1,2:Plasmids after BsmbI digesting; Lane3:Plasmids without BsmbI digestion (b)Recombinant plasmids were identified by PCR (c)Sequencing of recombinant lentiCRISPRv2

Table 1 sgRNA sequence

Fig.2 K562 cell line transfected by pX458 using Chemfect

Fig.3 The effect of different amount of transduced lentivirus on the survival of K562 cells under the treatment of purinomycin(parts)

Fig.4 Genetic mutation occurred around sgRNA3-1 trageting site in K562 cell pool transduced by lentivirus (a) Results from Sanger sequencing showing overlapping peaks occured around sgRNA3-1 targeting site (b) DNA sequence with different Indel mutations at sgRNA3-1 targeting site

Fig.5 Chromatography generated by Sanger sequencing showing the partial DNA sequence of monoclone B cell line around sgRNA3-1 targeting site (a) Results from Sanger sequencing shwoing overlapping peaks occured around sgRNA3-1 targeting site of JAK2 gene in monoclone B (b) Indel occurred in double allele DNA sequence around sgRNA3-1 targetting site of JAK2 gene in monoclone B The arrows represent Indel mutation

Fig.6 Functional verification experiments of K562-JAK2^-/-cell line (a) Identification of clone forming ability in methyl cellulose semisolid medium (b)The proliferation curve (CCK8) (c) K562 cells were induced to erythroid differentiation by Hemin and benzidine stained


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