Abstract
Interest is increasing in transcription activator-like effector nucleases (TALENs) as a tool to introduce targeted double-strand breaks into the large genomes of human and animal cell lines. The produced DNA lesions stimulate DNA repair pathways, error-prone but dominant non-homologous end joining (NHEJ) and accurate but less occurring homology-directed repair (HDR), and as a result targeted genes can be modified. Here, we describe a modified Golden-Gate cloning method for generating TALENs and also details for targeting genes in mouse embryonic stem cells. The protocol described here can be used for modifying the genome of a broad range of pluripotent cell lines.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ (2010) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29(2):143–148
Wood AJ, Lo T-W, Zeitler B, Pickle CS, Ralston EJ, Lee AH, Amora R, Miller JC, Leung E, Meng X (2011) Targeted genome editing across species using ZFNs and TALENs. Science 333(6040):307
Moore FE, Reyon D, Sander JD, Martinez SA, Blackburn JS, Khayter C, Ramirez CL, Joung JK, Langenau DM (2012) Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs). PLoS One 7(5):e37877
DeFrancesco L (2011) Move over ZFNs. Nat Biotechnol 29:681–684
Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31(2):294–301
Sander JD, Zaback P, Joung JK, Voytas DF, Dobbs D (2009) An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins. Nucleic Acids Res 37(2):506–515
Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39(12):e82
Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol 29(2):149–153
Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK (2012) FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30(5):460–465
Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F (2012) A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7(1):171–192
Schmid-Burgk JL, Schmidt T, Kaiser V, Höning K, Hornung V (2013) A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Nat Biotechnol 31:76–81
Perez-Pinera P, Ousterout DG, Brunger JM, Farin AM, Glass KA, Guilak F, Crawford GE, Hartemink AJ, Gersbach CA (2013) Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat Methods 10:239–242
Dahlem TJ, Hoshijima K, Jurynec MJ, Gunther D, Starker CG, Locke AS, Weis AM, Voytas DF, Grunwald DJ (2012) Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome. PLoS Genet 8(8):e1002861
Tong C, Huang G, Ashton C, Wu H, Yan H, Ying Q-L (2012) Rapid and cost-effective gene targeting in rat embryonic stem cells by TALENs. J Genet Genomics 39(6):275–280
Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG II, Tan W, Penheiter SG, Ma AC, Leung AY (2012) In vivo genome editing using a high-efficiency TALEN system. Nature 491(7422):114–118
Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, Voytas DF (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 161(1):20–27
Mashimo T, Kaneko T, Sakuma T, Kobayashi J, Kunihiro Y, Voigt B, Yamamoto T, Serikawa T (2013) Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes. Sci Rep 3:1253
Sakuma T, Hosoi S, Woltjen K, Suzuki K, Kashiwagi K, Wada H, Ochiai H, Miyamoto T, Kawai N, Sasakura Y (2013) Efficient TALEN construction and evaluation methods for human cell and animal applications. Genes Cells 18(4):315–326
Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, Voytas DF, Long CR, Whitelaw CBA, Fahrenkrug SC (2012) Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci U S A 109(43):17382–17387
Doyle EL, Booher NJ, Standage DS, Voytas DF, Brendel VP, VanDyk JK, Bogdanove AJ (2012) TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res 40(Web Server issue):W117–W122
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Taheri-Ghahfarokhi, A., Malaver-Ortega, L.F., Sumer, H. (2015). Genome Modification of Pluripotent Cells by Using Transcription Activator-Like Effector Nucleases (TALENs). In: Verma, P., Sumer, H. (eds) Cell Reprogramming. Methods in Molecular Biology, vol 1330. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2848-4_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2848-4_19
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2847-7
Online ISBN: 978-1-4939-2848-4
eBook Packages: Springer Protocols