Abstract
Genetic modification is an indispensable tool to study gene function in normal development and disease. The recent breakthrough of creating human induced pluripotent stem cells (iPSCs) by defined factors (Takahashi et al., Cell 131:861–872, 2007) provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the body including neurons and glia. Meanwhile, the rapid advancement of genome modification tools such as gene targeting by homologous recombination (Capecchi, Nat Rev Genet 6:507–512, 2005) and genome editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, TALENs (Transcription activator-like effector nucleases), and ZFNs (Zinc finger nucleases) (Wang et al., Cell 153:910–918, 2013; Mali et al., Science 339:823–826, 2013; Hwang et al., Nat Biotechnol 31:227–229, 2013; Friedland et al., Nat Methods 10(8):741–743, 2013; DiCarlo et al., Nucleic Acids Res 41:4336–4343, 2013; Cong et al., Science 339:819–823, 2013) has greatly accelerated the development of human genome manipulation at the molecular level. This chapter describes the protocols for making neural lineage reporter lines using homologous recombination and the CRISPR/Cas system-mediated genome editing, including construction of targeting vectors, guide RNAs, transfection into hPSCs, and selection and verification of successfully targeted clones. This method can be applied to various needs of hPSC genetic engineering at high efficiency and high reliability.
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Acknowledgments
This work was supported by the Department of Neurosurgery, Memorial Hermann Foundation-Staman Ogilvie Fund, the Bentsen Stroke Center, at the University of Texas Health Science center at Houston, Robert Packard Center for ALS Research at Johns Hopkins (Grant No. 2000260538), California Institute for Regenerative Medicine (Grant No. RT1-01107-1) to YL, and funds from the National Institutes of Health, Center for Regenerative Medicine to MSR. We thank Dr. J. Zhang for technical assistance.
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Xue, H., Wu, J., Li, S., Rao, M.S., Liu, Y. (2014). Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System. In: Turksen, K. (eds) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 1307. Humana Press, New York, NY. https://doi.org/10.1007/7651_2014_73
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DOI: https://doi.org/10.1007/7651_2014_73
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