Highly efficient genome editing via 2A-coupled co-expression of two TALEN monomers

BMC Research Notes

Renzhi Han²  Li Xu²  Andrew Mariano¹ 

[1] Department of Cell and Molecular Physiology, Loyola University Chicago Health Sciences Division, Maywood, IL 60153, USA;Department of Surgery, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA

关键词: 2A self-cleaving sequence;    TALEN;    Gene editing;   

Others  :  1129642
DOI  :  10.1186/1756-0500-7-628

 received in 2013-11-20, accepted in 2014-09-04,  发布年份 2014

【 摘 要 】

Background

Transcription activator-like effector nucleases (TALENs) are a useful tool for targeted gene editing. TALEN monomers are traditionally expressed from two different plasmids. Each encodes a different TALEN arm that binds to a user-defined sequence and mediates gene editing. Expression of TALEN monomers in two separate plasmids requires co-delivery of each plasmid to the cell. Efficacy of gene editing may be increased if each monomer was transcribed from the same reading frame.

Findings

We developed a TALEN scaffold which expresses both TALEN monomers from a single open reading frame in equal molar amount by linking both monomers with a 2A self-cleaving peptide sequence. This TALEN scaffold, named pTAL10, demonstrates higher levels of genome editing than co-transfected TALENs at similar levels of transfection efficiencies when analyzed for TALEN-induced small insertions and deletions.

Conclusions

This protocol for gene editing using 2A-linked TALENs requires transfection of only one plasmid as compared to transfection of two separate plasmids encoding each TALEN monomers.

【 授权许可】

   
2014 Mariano et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T: A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 2011, 39(21):9283-9293.
• [2]Li T, Huang S, Zhao X, Wright DA, Carpenter S, Spalding MH, Weeks DP, Yang B: Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res 2011, 39(14):6315-6325.
• [3]Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng X, Hinkley SJ, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R: Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 2011, 29(8):731-734.
• [4]Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF: Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 2011, 39(12):e82.
• [5]Moscou MJ, Bogdanove AJ: A simple cipher governs DNA recognition by TAL effectors. Science 2009, 326(5959):1501.
• [6]Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U: Breaking the code of DNA binding specificity of TAL-type III effectors. Science 2009, 326(5959):1509-1512.
• [7]Doyle EL, Booher NJ, Standage DS, Voytas DF, Brendel VP, Vandyk JK, Bogdanove AJ: TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res 2012, 40:W117-W122. Web Server issue
• [8]Joung JK, Sander JD: TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 2013, 14(1):49-55.
• [9]Guo J, Gaj T, Barbas CF 3rd: Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. J Mol Biol 2010, 400(1):96-107.
• [10]Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ: A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 2011, 29(2):143-148.
• [11]Sun N, Bao Z, Xiong X, Zhao H: SunnyTALEN: a second-generation TALEN system for human genome editing. Biotechnol Bioeng 2014, 111(4):683-691.
• [12]Duda K, Lonowski LA, Kofoed-Nielsen M, Ibarra A, Delay CM, Kang Q, Yang Z, Pruett-Miller SM, Bennett EP, Wandall HH, Davis GD, Hansen SH, Frödin M: High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs. Nucleic Acids Res 2014, 42(10):e84.
• [13]Xu L, Zhao P, Mariano A, Han R: Targeted myostatin gene editing in multiple mammalian species directed by a single pair of TALE nucleases. Mol Ther Nucleic Acids 2013, 2:e112.
• [14]Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, Vignali DA: Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 2004, 22(5):589-594.
• [15]Donnelly ML, Hughes LE, Luke G, Mendoza H, ten Dam E, Gani D, Ryan MD: The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences. J Gen Virol 2001, 82(Pt 5):1027-1041.
• [16]Park SH, Chung HK, Kim Do J, Han MR, Park MS, Oh U, Kim HJ, Han BW: Overexpression, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytosolic domain of mouse anoctamin 1. Acta Crystallogr Sect F Struct Biol Cryst Commun 2011, 67(Pt 10):1250-1252.
• [17]Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ: An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 2007, 25(7):778-785.
• [18]Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG 2nd, Tan W, Penheiter SG, Ma AC, Leung AY, Fahrenkrug SC, Carlson DF, Voytas DF, Clark KJ, Essner JJ, Ekker SC: In vivo genome editing using a high-efficiency TALEN system. Nature 2012, 491(7422):114-118.