Abstract
Genome engineering and human iPS cells are two powerful technologies, which can be combined to highlight phenotypic differences and identify pathological mechanisms of complex diseases by providing isogenic cellular material. However, very few data are available regarding precise gene correction in human iPS cells. Here, we describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. We highlight technical issues especially those associated to human stem cell culture and to the correction of a point mutation to obtain isogenic iPS cell line, without inserting any resistance cassette. Based on a two-steps clonal isolation protocol (mechanical picking followed by enzymatic dissociation), we succeed to select and expand corrected human iPS cell line with a great efficiency (more than 2 % of the sequenced colonies). This protocol can also be used to obtain knock-out cell line from healthy iPS cell line by the NHEJ pathway (with about 15 % efficiency) and reproduce disease phenotype. In addition, we also provide protocols for functional validation tests after every critical step.
Similar content being viewed by others
References
Sebat, J., Levy, D. L., & McCarthy, S. E. (2009). Rare structural variants in schizophrenia: one disorder, multiple mutations; one mutation, multiple disorders. Trends in Genetics, 25, 528–535.
Dipple, K. M., & McCabe, E. R. (2000). Phenotypes of patients with “simple” Mendelian disorders are complex traits: thresholds, modifiers, and systems dynamics. American Journal of Human Genetics, 66, 1729–1735.
Musunuru, K. (2013). Genome editing of human pluripotent stem cells to generate human cellular disease models. Disease Models & Mechanisms, 6, 896–904.
Kim, H., Jang, M. J., Kang, M. J., & Han, Y. M. (2011). Epigenetic signatures and temporal expression of lineage-specific genes in hESCs during differentiation to hepatocytes in vitro. Human Molecular Genetics, 20, 401–412.
Lister, R., Pelizzola, M., Kida, Y. S., et al. (2011). Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature, 471, 68–73.
Ruiz, S., Diep, D., Gore, A., et al. (2012). Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America, 109, 16196–16201.
Zwaka, T. P., & Thomson, J. A. (2003). Homologous recombination in human embryonic stem cells. Nature Biotechnology, 21, 319–321.
Placantonakis, D. G., Tomishima, M. J., Lafaille, F., et al. (2009). BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage. Stem Cells, 27, 521–532.
Song, H., Chung, S. K., & Xu, Y. (2010). Modeling disease in human ESCs using an efficient BAC-based homologous recombination system. Cell Stem Cell, 6, 80–89.
Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., & Doudna, J. (2013). RNA-programmed genome editing in human cells. eLife, 2, e00471.
Cong, L., Ran, F. A., Cox, D., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science (New York, NY), 339, 819–823.
Mali, P., Yang, L., Esvelt, K. M., et al. (2013). RNA-guided human genome engineering via Cas9. Science (New York, NY), 339, 823–826.
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (New York, NY), 337, 816–821.
Lin, S., Staahl, B. T., Alla, R. K., & Doudna, J. A. (2014). Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife, 3, e04766.
Chen, F., Pruett-Miller, S. M., Huang, Y., et al. (2011). High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nature Methods, 8, 753–755.
Doench, J. G., Hartenian, E., Graham, D. B., et al. (2014). Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nature Biotechnology, 32, 1262–1267.
Huang, X., Wang, Y., Yan, W., et al. (2015). Production of gene-corrected adult beta globin protein in human erythrocytes differentiated from patient iPSCs after genome editing of the sickle point mutation. Stem Cells, 33, 1470–1479.
Li, H. L., Fujimoto, N., Sasakawa, N., et al. (2015). Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Reports, 4, 143–154.
Miyaoka, Y., Chan, A. H., Judge, L. M., et al. (2014). Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nature Methods, 11, 291–293.
Reinhardt, P., Schmid, B., Burbulla, L. F., et al. (2013). Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegeneration to ERK-dependent changes in gene expression. Cell Stem Cell, 12, 354–367.
Soldner, F., Laganiere, J., Cheng, A. W., et al. (2011). Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell, 146, 318–331.
Ye, L., Wang, J., Beyer, A. I., et al. (2014). Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Delta32 mutation confers resistance to HIV infection. Proceedings of the National Academy of Sciences of the United States of America, 111, 9591–9596.
Yusa, K. (2013). Seamless genome editing in human pluripotent stem cells using custom endonuclease-based gene targeting and the piggyBac transposon. Nature Protocols, 8, 2061–2078.
Wen, Z., Nguyen, H. N., Guo, Z., et al. (2014). Synaptic dysregulation in a human iPS cell model of mental disorders. Nature, 515, 414–418.
Guschin, D. Y., Waite, A. J., Katibah, G. E., Miller, J. C., Holmes, M. C., & Rebar, E. J. (2010). A rapid and general assay for monitoring endogenous gene modification. Methods in Molecular Biology, 649, 247–256.
Froger, A., Hall, J. E. (2007). Transformation of plasmid DNA into E. coli using the heat shock method. Journal of Visualized Experiments: JoVE, 253.
Vouillot, L., Thelie, A., & Pollet, N. (2015). Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3, 5, 407–415.
Smithies, O., Gregg, R. G., Boggs, S. S., Koralewski, M. A., & Kucherlapati, R. S. (1985). Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature, 317, 230–234.
Hasty, P., Rivera-Perez, J., & Bradley, A. (1991). The length of homology required for gene targeting in embryonic stem cells. Molecular and Cellular Biology, 11, 5586–5591.
Lin, Y., Cradick, T. J., Brown, M. T., et al. (2014). CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research, 42, 7473–7485.
Takahashi, K., Tanabe, K., Ohnuki, M., et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–872.
Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012). Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 109, E2579–E2586.
Ran, F. A., Hsu, P. D., Lin, C. Y., et al. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 154, 1380–1389.
Conflict of Interest
The authors indicate no potential conflicts of interest.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Grobarczyk, B., Franco, B., Hanon, K. et al. Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System. Stem Cell Rev and Rep 11, 774–787 (2015). https://doi.org/10.1007/s12015-015-9600-1
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12015-015-9600-1