title: Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9.
abstract: 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.
authors.affiliation: Waisman Center, University of Wisconsin, Madison, WI 53705, USA. Electronic address: yuejunchenphd@gmail.com.
authors.forename: Yuejun
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Waisman Center, University of Wisconsin, Madison, WI 53705, USA.
authors.forename: Jingyuan
authors.initials: J
authors.lastname: Cao
authors.affiliation: Waisman Center, University of Wisconsin, Madison, WI 53705, USA; Institute of Pediatrics, Children's Hospital, Fudan University, 399 Wanyuan Road, Shanghai 201102, China.
authors.forename: Man
authors.initials: M
authors.lastname: Xiong
authors.affiliation: Waisman Center, University of Wisconsin, Madison, WI 53705, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Petersen
authors.affiliation: Waisman Center, University of Wisconsin, Madison, WI 53705, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Waisman Center, University of Wisconsin, Madison, WI 53705, USA.
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Tao
authors.affiliation: Waisman Center, University of Wisconsin, Madison, WI 53705, USA.
authors.forename: Cindy Tzu-Ling
authors.initials: CT
authors.lastname: Huang
authors.affiliation: Waisman Center, University of Wisconsin, Madison, WI 53705, USA.
authors.forename: Zhongwei
authors.initials: Z
authors.lastname: Du
authors.affiliation: 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.
authors.forename: Su-Chun
authors.initials: SC
authors.lastname: Zhang
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002454:Cell Differentiation; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D006720:Homozygote; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D051857:Otx Transcription Factors / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism*
chemical_list: C497659:OTX2 protein, human; D051857:Otx Transcription Factors; D049294:Phospholipid Transfer Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems