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Nucleic Acids Res. 2014 Mar;42(5):e34. doi: 10.1093/nar/gkt1290. Epub 2013 Dec 4.

DICE, an efficient system for iterative genomic editing in human pluripotent stem cells.

Author information

1
Department of Genetics, Stanford University, Stanford, CA 94305, USA, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA, Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, CA 94305, USA, Parkinson's Institute and Clinical Center, Sunnyvale, CA 94085, USA and Stanford Transgenic Research Facility, Stanford University, Stanford, CA 94305, USA.

Abstract

To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11, located in a safe, intergenic, transcriptionally active region of chromosome 22, as the recipient site, to provide robust, ubiquitous expression of inserted genes. Recipient cell lines were established by site-specific placement of a 'landing pad' cassette carrying attP sites for phiC31 and Bxb1 integrases at the H11 locus by spontaneous or TALEN-assisted homologous recombination. Dual integrase cassette exchange (DICE) mediated by phiC31 and Bxb1 integrases was used to insert genes of interest flanked by phiC31 and Bxb1 attB sites at the H11 locus, replacing the landing pad. This system provided complete control over content, direction and copy number of inserted genes, with a specificity of 100%. A series of genes, including mCherry and various combinations of the neural transcription factors LMX1a, FOXA2 and OTX2, were inserted in recipient cell lines derived from H9 ESC, as well as iPSC lines derived from a Parkinson's disease patient and a normal sibling control. The DICE system offers rapid, efficient and precise gene insertion in ESC and iPSC and is particularly well suited for repeated modifications of the same locus.

PMID:
24304893
PMCID:
PMC3950688
DOI:
10.1093/nar/gkt1290
[Indexed for MEDLINE]
Free PMC Article

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