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The Establishment of Inducible MEIS1 Knockout HEK293T Cell Line by CRISPR/Cas9
Zhang Mingzhi1#, Wang Mengge2#, Wang Hongtao2, Su Pei2, Liu Xin2, Zhang Leisheng2, Liu Cuicui2, Wang Yu2, Wu Dan2, Zhou Jiaxi2*, Peng Sha1*
1College of Veterinary Medicine, Shaanxi Stem Cell Engineering and Technology Research Center, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest Agriculture and Forestry University, Yangling 712100, China;
2Stata Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China
2Stata Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China
Abstract: CRISPR/Cas9 is a novel genome editing tool which enables rapid and highly efficient generation of biallelic knockout cell lines for loss-of-function studies， as well as homozygous knockin cell lines with specific nucleotide alterations for disease modeling. However， there are lots of limitations in common CRISPR/Cas9 system，such as low efficiency of direct transfection and virus packaging. In this study， we used a Tet-on system to establish an inducible genome-engineering platform which can express the Cas9 with Doxychline treatment in HEK293T cell line named as 293T-iCas9 cell line. MEIS1， a transcription factor of TALE homeodomain family， plays important roles in leukemia， hematopoiesis and the nervous development. However， the mechanism by which MEIS1 regulates these processes remains unclear. We transfected a sgRNA expression vector targeting the Exon3 of MEIS1 into 293T-iCas9 cell line. SURVEVOR assay and Western blot results demonstrated that sgMEIS1 was able to edit genomic DNA at MEIS1 loci. MEIS1 knockout cell line had been derived successfully after further confirmation by sequecing and Western blot. It could be used as an important tool for futher study of the function of MEIS1.
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