Abstract
Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double strand breaks (DSB) at defined sequences in the human genome. These systems typically use plasmids to transiently transcribe nucleases within the cell. Here, we describe the process for preparing hPSCs for transient expression of nucleases via electroporation and subsequent analysis to create genetically modified stem cell lines.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sonoda E, Hochegger H, Saberi A et al (2006) Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair 5:1021–1029
Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Chen F, Pruett-Miller SM, Huang Y et al (2011) High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Methods 8:753–755
Hockemeyer D, Wang H, Kiani S et al (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29:731–734
Zwaka TP, Thomson JA (2003) Homologous recombination in human embryonic stem cells. Nat Biotechnol 21:319–321
Ran FA, Hsu PD, Wright J et al (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308
González F, Zhu Z, Shi Z-D et al (2004) An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15(2):215–226
Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11:783–784
Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84
Shalem O, Sanjana NE, Hartenian E et al (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84–87
Lakshmipathy U, Pelacho B, Sudo K et al (2004) Efficient transfection of embryonic and adult stem cells. Stem Cells 22:531–543
Ding Q, Regan S, Xia Y et al (2013) Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12:393–394
Cordie T, Harkness T, Jing X et al (2014) Nanofibrous electrospun polymers for reprogramming human cells. Cell Mol Bioeng 7:379–393
Qiu P (2004) Mutation detection using SurveyorTM nuclease. Biotechniques 36:702–707
Zhu X, Xu Y, Yu S et al (2014) An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system. Sci Rep 4:6420
Caporaso JG, Lauber CL, Walters WA et al (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6:1621–1624
Wittwer CT, Reed GH, Gundry CN et al (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853–860
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci 74:5463–5467
Cho SW, Kim S, Kim JM, Kim J-S (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232
Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA—guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Hwang WY, Fu Y, Reyon D et al (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229
Yang L, Guell M, Byrne S et al (2013) Optimization of scarless human stem cell genome editing. Nucleic Acids Res 41(19):9049–9061
Fu Y, Sander JD, Reyon D et al (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32:279–284
Hockemeyer D, Soldner F, Beard C et al (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27:851–857
Miyaoka Y, Chan AH, Judge LM et al (2014) Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat Methods 11:291–229
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Carlson-Stevermer, J., Saha, K. (2017). Genome Editing in Human Pluripotent Stem Cells. In: Crook, J., Ludwig, T. (eds) Stem Cell Banking. Methods in Molecular Biology, vol 1590. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6921-0_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6921-0_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6919-7
Online ISBN: 978-1-4939-6921-0
eBook Packages: Springer Protocols