DICE, an Efficient System for Iterative Genomic Editing in Human Pluripotent Stem Cells

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2013 Dec 6

PMID 24304893

Citations 54

Authors

Fangfang Zhu

Matthew Gamboa

Alfonso P Farruggio

Simon Hippenmeyer

Bosiljka Tasic

Birgitt Schule

Yanru Chen-Tsai

Michele P Calos

Affiliations

Soon will be listed here.

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.

Citing Articles

There and turn back again: the application of phage serine integrases in eukaryotic systems.

Sales T, de Oliveira M, Florentino L, Lima R, Rech E Front Bioeng Biotechnol. 2025; 13:1478413.

PMID: 40066361 PMC: 11891168. DOI: 10.3389/fbioe.2025.1478413.

Expression of transgenic biotin ligases in inducible neuronal murine cell lines by integration into the mHipp11 gene locus.

Feicht L, Dangel A, Jansen R PLoS One. 2025; 20(3):e0315806.

PMID: 40036200 PMC: 11878913. DOI: 10.1371/journal.pone.0315806.

Ubiquitous MEIS transcription factors actuate lineage-specific transcription to establish cell fate.

Darieva Z, Zarrineh P, Phillips N, Mallen J, Garcia Mora A, Donaldson I EMBO J. 2025; .

PMID: 40021842 DOI: 10.1038/s44318-025-00385-5.

Multiplex, multimodal mapping of variant effects in secreted proteins.

Popp N, Powell R, Wheelock M, Holmes K, Zapp B, Sheldon K bioRxiv. 2025; .

PMID: 39975210 PMC: 11838247. DOI: 10.1101/2024.04.01.587474.

C-terminal tagging, transmembrane domain hydrophobicity, and an ER retention motif influence the secretory trafficking of the inner nuclear membrane protein emerin.

Mella J, Volk R, Zaro B, Buchwalter A bioRxiv. 2025; .

PMID: 39896633 PMC: 11785080. DOI: 10.1101/2025.01.19.633811.

References

Carey B, Markoulaki S, Hanna J, Saha K, Gao Q, Mitalipova M . Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc Natl Acad Sci U S A. 2008; 106(1):157-62. PMC: 2629226. DOI: 10.1073/pnas.0811426106. View

Friling S, Andersson E, Thompson L, Jonsson M, Hebsgaard J, Nanou E . Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells. Proc Natl Acad Sci U S A. 2009; 106(18):7613-8. PMC: 2671325. DOI: 10.1073/pnas.0902396106. View

Shulman J, De Jager P, Feany M . Parkinson's disease: genetics and pathogenesis. Annu Rev Pathol. 2010; 6:193-222. DOI: 10.1146/annurev-pathol-011110-130242. View

Leavitt A, Hamlett I . Homologous recombination in human embryonic stem cells: a tool for advancing cell therapy and understanding and treating human disease. Clin Transl Sci. 2011; 4(4):298-305. PMC: 5439769. DOI: 10.1111/j.1752-8062.2011.00281.x. View

Belteki G, Gertsenstein M, Ow D, Nagy A . Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase. Nat Biotechnol. 2003; 21(3):321-4. DOI: 10.1038/nbt787. View

Ni W, Hu S, Qiao J, Wang Y, Shi H, Wang Y . ΦC31 integrase mediates efficient site-specific integration in sheep fibroblasts. Biosci Biotechnol Biochem. 2012; 76(11):2093-5. DOI: 10.1271/bbb.120439. View

Lin W, Metzakopian E, Mavromatakis Y, Gao N, Balaskas N, Sasaki H . Foxa1 and Foxa2 function both upstream of and cooperatively with Lmx1a and Lmx1b in a feedforward loop promoting mesodiencephalic dopaminergic neuron development. Dev Biol. 2009; 333(2):386-96. DOI: 10.1016/j.ydbio.2009.07.006. View

Chen C, Krohn J, Bhattacharya S, Davies B . A comparison of exogenous promoter activity at the ROSA26 locus using a ΦiC31 integrase mediated cassette exchange approach in mouse ES cells. PLoS One. 2011; 6(8):e23376. PMC: 3154917. DOI: 10.1371/journal.pone.0023376. View

Hippenmeyer S, Youn Y, Moon H, Miyamichi K, Zong H, Wynshaw-Boris A . Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration. Neuron. 2010; 68(4):695-709. PMC: 3044607. DOI: 10.1016/j.neuron.2010.09.027. View

10.

Mali P, Yang L, Esvelt K, Aach J, Guell M, DiCarlo J . RNA-guided human genome engineering via Cas9. Science. 2013; 339(6121):823-6. PMC: 3712628. DOI: 10.1126/science.1232033. View

11.

Chung S, Leung A, Han B, Chang M, Moon J, Kim C . Wnt1-lmx1a forms a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway. Cell Stem Cell. 2009; 5(6):646-58. PMC: 2788512. DOI: 10.1016/j.stem.2009.09.015. View

12.

Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S . Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009; 326(5959):1509-12. DOI: 10.1126/science.1178811. View

13.

Szymczak A, Workman C, Wang Y, Vignali K, Dilioglou S, Vanin E . Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat Biotechnol. 2004; 22(5):589-94. DOI: 10.1038/nbt957. View

14.

Bateman J, Lee A, Wu C . Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange. Genetics. 2006; 173(2):769-77. PMC: 1526508. DOI: 10.1534/genetics.106.056945. View

15.

Venken K, He Y, Hoskins R, Bellen H . P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science. 2006; 314(5806):1747-51. DOI: 10.1126/science.1134426. View

16.

Pan H, Zhang W, Zhang W, Liu G . Find and replace: editing human genome in pluripotent stem cells. Protein Cell. 2011; 2(12):950-6. PMC: 4875250. DOI: 10.1007/s13238-011-1132-0. View

17.

Keravala A, Groth A, Jarrahian S, Thyagarajan B, Hoyt J, Kirby P . A diversity of serine phage integrases mediate site-specific recombination in mammalian cells. Mol Genet Genomics. 2006; 276(2):135-46. DOI: 10.1007/s00438-006-0129-5. View

18.

Michael I, Monetti C, Chiu A, Zhang P, Baba T, Nishino K . Highly efficient site-specific transgenesis in cancer cell lines. Mol Cancer. 2012; 11:89. PMC: 3537676. DOI: 10.1186/1476-4598-11-89. View

19.

cermak T, Doyle E, Christian M, Wang L, Zhang Y, Schmidt C . Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011; 39(12):e82. PMC: 3130291. DOI: 10.1093/nar/gkr218. View

20.

Tasic B, Miyamichi K, Hippenmeyer S, Dani V, Zeng H, Joo W . Extensions of MADM (mosaic analysis with double markers) in mice. PLoS One. 2012; 7(3):e33332. PMC: 3314016. DOI: 10.1371/journal.pone.0033332. View