Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9.

Autor:	Chen Y; Waisman Center, University of Wisconsin, Madison, WI 53705, USA. Electronic address: yuejunchenphd@gmail.com., Cao J; Waisman Center, University of Wisconsin, Madison, WI 53705, USA., Xiong M; Waisman Center, University of Wisconsin, Madison, WI 53705, USA; Institute of Pediatrics, Children's Hospital, Fudan University, 399 Wanyuan Road, Shanghai 201102, China., Petersen AJ; Waisman Center, University of Wisconsin, Madison, WI 53705, USA., Dong Y; Waisman Center, University of Wisconsin, Madison, WI 53705, USA., Tao Y; Waisman Center, University of Wisconsin, Madison, WI 53705, USA., Huang CT; Waisman Center, University of Wisconsin, Madison, WI 53705, USA., Du Z; Waisman Center, University of Wisconsin, Madison, WI 53705, USA., Zhang SC; Waisman Center, University of Wisconsin, Madison, WI 53705, USA; Neuroscience Training Program, University of Wisconsin, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705, USA; Department of Neurology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705, USA. Electronic address: zhang@waisman.wisc.edu.
Jazyk:	angličtina
Zdroj:	Cell stem cell [Cell Stem Cell] 2015 Aug 06; Vol. 17 (2), pp. 233-44. Date of Electronic Publication: 2015 Jul 02.
DOI:	10.1016/j.stem.2015.06.001
Abstrakt:	Precise temporal control of gene expression or deletion is critical for elucidating gene function in biological systems. However, the establishment of human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) remains challenging. We explored building iKO hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system. We found that "dual-sgRNA targeting" is essential for biallelic knockin of FRT sequences to flank the exon. We further developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene, thus speeding up the generation of iKO hPSC lines. This two-step strategy was used to establish human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines with iKO of SOX2, PAX6, OTX2, and AGO2, genes that exhibit diverse structural layout and temporal expression patterns. The availability of iKO hPSC lines will substantially transform the way we examine gene function in human cells. (Copyright © 2015 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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