- Open Access
Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs
- Renli Ru†1,
- Yongchao Yao†1,
- Songlin Yu1,
- Benpeng Yin1,
- Wanwan Xu1,
- Siting Zhao1,
- Li Qin1 and
- Xiaoping Chen1, 2Email author
© Ru et al.; licensee BioMed Central Ltd. 2013
Received: 26 March 2013
Accepted: 3 June 2013
Published: 18 June 2013
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However, the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.
In this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN, respectively. However, TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5, a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.
TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology.
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are two types of artificial site-specific nucleases that are generated by fusing the DNA cleavage domain of the FokI restriction endonuclease with a custom-designed DNA biding domain. In ZFNs and TALENs, these DNA-binding domains are the C2H2 zinc-finger motif  and the transcription activator-like effector (TALE) domain respectively . Because the FokI nuclease domain functions as a dimer, ZFNs and TALENs are typically used in pairs. These chimeric nucleases induce DNA double-strand breaks at defined sites in living cells that can be repaired via homology-directed repair (HDR) pathway, which utilizes a homologous donor DNA, or imprecise nonhomologous end joining (NHEJ) pathway . These engineered nucleases have been widely used to modify endogenous genes in human stem cells [4–7]. However, gene-based delivery systems are problematic. In particular, the use of viral vectors entails the risk of insertional mutagenesis , whereas plasmid DNA has cell-type restrictions and cytotoxicity.
Cell-penetrating peptides (CPPs), also known as protein transduction domain (PTD) peptides, have been used to deliver a variety of cargoes (including drug molecules, nucleic acids, liposome nanoparticles and large proteins) into cells both in vivo and in vitro and have therefore shown great promise as therapeutic delivery mechanisms for the treatment of several diseases . The CPP delivery system has various advantages including applicability to all cell types, high transduction efficiency and controlled administration. Among the CPPs, TAT (YGRKKRRQRRR) is the most widely investigated and widely used peptide. TAT is a short peptide consisting of 11 amino acids from the human immunodeficiency virus-1 (HIV-1) TAT protein and is rich in arginine and lysine; therefore, TAT is highly positively charged and hydrophilic . Since the first use of TAT as a delivery vehicle for introducing molecules into cells in the late 1990s, numerous studies have reported the use of TAT for the delivery of various biomolecules (especially proteins) into cells in the form of TAT fusion proteins or TAT-protein conjugates . Use of TAT as a delivery vehicle has been considered as one of the most promising gene-free strategies.
In this study, we designed TAT-ZFN and TAT-TALEN fusion proteins by in-frame cloning. We succeeded in purifying functional TAT-TALEN proteins, and demonstrated their cell penetrating properties. When incubated with living cells, TAT-TALENs efficiently disrupted the endogenous CCR5 gene under hypothermic conditions in a dose-dependent manner and we observed a disruption efficiency of up to 5% in human induced pluripotent stem cells (hiPSCs).
Purification and activity testing of TAT-ZFNs and TAT-TALENs
Transduction of proteins into living cells
CCR5 disruption in HeLa cells and hiPSCs
To explore the potential of this method for practical application, we evaluated the effects of CCR5 gene disruption via TAT-TALEN proteins in hiPSCs. Under hypothermic conditions, hiPSCs subjected to three consecutive treatments with 3 μM TAT-TALEN proteins showed gene disruption frequencies of up to 5% (Figure 5A). It is unknown whether cell membrane composition or endocytosis capabilities could explain the observed differences between HeLa cells and hiPSCs with regard to CCR5 gene disruption frequency.
TAT-TALEN cytotoxicity analysis
Targeted genomic engineering in stem cells at desired endogenous gene loci by ZFNs or TALENs has significant therapeutic implications . The correction of disease-causing mutations in patient-derived stem cells is a goal of curative regenerative medicine. The disruption of a normal gene in stem cells can also be beneficial for the treatment of certain diseases. CCR5 is a major co-receptor for HIV-1 that is present on the surface of target cells . Currently, autologous transplantation of CCR5-deficient human hematopoietic stem cells (hHSCs) has been a promising strategy for acquired immunodeficiency syndrome (AIDS) treatment [13–17]. Notably, HIV-1 can establish latent viral reservoirs in hHSCs  prior to genome engineering; thus, caution must be exercised when using these cells. Our lab has successfully disrupted the CCR5 locus of hiPSCs using ZFNs and demonstrated that these CCR5-disrupted hiPSCs can differentiate into CD34+ cells with multipotency in vitro . This work contributes to the advancement of patient-specific therapies with genetically modified hiPSCs produced from the healthy tissues of HIV-infected patients. However, some problems such as safety concerns regarding the gene-based delivery method of ZFNs and TALENs need to be addressed [8, 20]. In addition to mRNA delivery system, our TAT-TALEN protein provides an alternative to eliminate the risk of insertional mutagenesis and expands the range of cell types that the nucleases can be applied to modify.
The concept of CPP-ZFN proteins has been proposed previously [21, 22]. However, we failed to utilize TAT-ZFN to modify the endogenous gene because of a purification problem. Recently, a study by Gaj and colleagues demonstrated the intrinsic cell-penetrating capabilities of standard ZFN proteins and their ability to successfully modify CCR5 in living cells . Compared with ZFNs, TALENs are more mutagenic  and more specific ; additionally, the design and assemble of TALENs is very simple and straightforward [26, 27]. In all these aspects, TALENs are superior to ZFNs.
We utilized TAT-TALEN to successfully disrupt endogenous CCR5 in HeLa cells and hiPSCs. It has been reported that hypothermia treatment improves ZFN-driven  and TALEN-driven  gene disruption in human cells transfected with eukaryotic expression vectors. We found that hypothermic treatment was also effective in cells transduced with TAT-TALENs. A high TALEN concentration may lead to an increase in off-target cleavage events in cells; cells treated with excessive amounts of TAT-TALENs are likely to die as a result of non-specific genome cleavage. This effect may account for the moderate cytotoxicity observed in this experiment. In our study, TAT was fused to the N-terminus of TALEN. It is unknown whether shifting the TAT peptide to the C- terminus of TALEN or using other CPPs such as polyarginine could further enhance protein penetration efficiency; however, similar adjustments need to be considered in future studies.
In summary, we generated functional TAT-TALEN proteins that were capable of penetrating cells. These constructs were used to efficiently disrupt CCR5 in HeLa cells and hiPSCs. Combined with our previous work, this gene-free delivery system has great potential for the clinical application of CCR5-disrupted hiPSCs. In addition, this new TALEN delivery method may be applied to the gene therapy for several monogenic diseases as well, a circumstance in which a DNA donor template is needed.
Construction of prokaryotic expression plasmids
ZFN mammalian expression vectors were previously constructed in our lab . TALEN mammalian expression vectors were constructed as described [26, 29]. The corresponding prokaryotic expression plasmids were constructed as follows: encoding sequences of ZFN and TALEN were cloned from their mammalian expression vectors. TAT-encoding sequences were introduced by primer design. All primer sequences are provided in Additional file 1: Table S1. The PCR products were digested with NdeI and BamHI for (ZFN) or NdeI and HindIII (for TALEN), and the digested products were inserted into pET28a (Novagen) to yield the following six plasmids: pET.TAT-ZFNL, pET.TAT-ZFNR, pET.TAT-TALENL, pET.TAT-TALENR, pET.TALENL, pET.TALENR. L and R stand for left and right, respectively. Left ZFN (or TALEN) and right ZFN (or TALEN) constitute a functional ZFN (or TALEN) pair. Proper construction of each expression cassette was verified by sequence analysis. The amino acid sequences of each protein are provided in Additional file 1: Table S2.
Expression and purification of fusion proteins
Recombinant expression plasmids were transformed into competent Escherichia-coli BL21 (DE3) cells. LB medium (40 ml) supplemented with 100 μg/ml kanamycin was inoculated with a single colony and incubated overnight at 37°C with shaking. On the following day, the overnight culture was diluted 50-fold into fresh LB medium supplemented with 100 μg/ml kanamycin and incubated at 37°C with shaking until the optical density at 600 nm (OD600) reached 0.5. The culture was then incubated at 16°C for 1 hour. Protein synthesis was induced by the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.7 mM, and the culture was grown overnight at 16°C with shaking. The cells were harvested and lysed five times using a French press (JN-3000 PLUS, JNBIO) at 10,000 psi in lysis buffer (25 mM Tris–HCl, pH 8.0, 300 mM NaCl, 5% V/V glycerol) at 4°C. After the cell debris was removed by centrifugation at 20,000 g for 30 minutes, TALEN proteins in the soluble fraction were purified using Ni-NTA agarose resin (QIAGEN) and eluted with elution buffer (lysis buffer with 500 mM imidazole, pH 8.0). For TAT-ZFN expression and purification, 100 μM ZnCl2 was added to all solutions.
Each protein was desalted using PD-10 columns (GE Healthcare) into PBS (pH 7.4) with 20% glycerol and sterilized by filtration through a 0.22 μM filter. The protein was then aliquoted and stored at −80°C. Protein purity was assessed by SDS-PAGE, and the total protein concentration was determined using a BCA protein assay kit (Beyotime, China). The total protein concentrations of standard TALEN and TAT-TALEN were between 0.9 and 1.2 mg/ml and between 0.3 and 0.5 mg/ml, respectively.
In vitro activity of purified proteins
Because the recognition sites of ZFN and TALEN are distinct from each other, CCR5 loci were amplified separately from the human genomic DNA with different primer pairs (Primer Pair 1 for ZFNs and Primer Pair 2 for TALENs). The substrate DNA was incubated with ZFN or TALEN in NEB buffer 4 for 1 hour at 37°C . Cleavage products were separated by agarose gel electrophoresis (1.5%) or polyacrylamide gel electrophoresis (8%) in 1 × TBE buffer and stained with ethidium bromide solution.
HeLa cells were seeded onto 48-well plates at a density of 5 × 104 cells per well and cultured in a humidified atmosphere containing 5% CO2 at 37°C. Cells were maintained in high-glucose DMEM (HyClone) containing 10% fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin.
hiPSCs were induced from the urine cells of a healthy individual using episomal vectors. Initial populations were seeded onto 48-well plates at the proper density and grown until cell confluence reached 60-70%. These cells were maintained in a feeder-free culture system using mTesR1 (Stem Cell Technologies, Vancouver, BC, Canada) and Matrigel (BD Bioscience, Bedford, MA) with 1% (v/v) penicillin-streptomycin.
TALEN proteins, including standard TALENs and TAT-TALENs were prepared for treatment as follows: TALEN proteins were diluted into serum-free high-glucose DMEM. The cells were washed with serum-free medium and incubated with TALEN proteins for 1 hour at 37°C. After the initial treatment, the cells were washed and maintained at 37°C or shifted to 30°C for 24 hours before the next treatment. This process was repeated three times over four consecutive days.
Western blot analysis to determine protein internalization
HeLa cells were treated with either 1.5 μM TALEN or 1.5 μM TAT-TALEN for 1 hour at 37°C and then washed three times with 1 mg/ml heparin-PBS to remove proteins bound to the cell surface. After trypsinization, the cells were collected and lysed in RIPA lysis buffer (Beyotime) and subsequently boiled in the presence of loading buffer (50 mM Tris–HCl, pH 6.8, 100 mM dithiothreitol, 2% sodium dodecyl sulfate (SDS), 20% glycerol and 0.2 mg/mL bromophenol blue) for 10 minutes and subjected to electrophoresis using 10% SDS-PAGE. The gel was transferred to a polyvinylidene difluoride membranes using a Trans-blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA). TALENs were verified using monoclonal anti-FLAG M2 antibodies (Sigma, 1:1000 dilution) and secondary HRP-labeled goat anti-mouse antibodies (Sigma, 1:5000 dilution). The internal loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was detected with peroxidase-conjugated anti-GAPDH antibody. The western blots were developed with DAB substrate according to the manufacturer’s instructions.
Surveyor nuclease assay
Genomic DNA was extracted from TALEN-treated cells with a DNeasy Blood and Tissue Kit (QIAGEN). The CCR5 locus was amplified by nested PCR, and the sequences of the outer and inner primers are provided in Additional file 1: Table S1. After amplification of the CCR5 locus using Platinum® Taq DNA Polymerase (Invitrogen), a Surveyor nuclease assay was performed following the instructions included with the SURVEYOR Mutation Detection Kit (Transgenomic). Analysis of the gene disruption frequency was performed as described . The gel image was processed with the ImageJ software.
Cell proliferation assay
HeLa cells and hiPSCs were seeded onto 48-well plates at the optimal densities. The cells were then treated with TAT-TALEN proteins as described above. Cytotoxicity assays were conducted using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.) according to the manufacturer’s instructions.
Genomic DNA from TAT-TALEN-treated HeLa cells was isolated with the DNeasy Blood and Tissue Kit (QIAGEN). The CCR5 locus was amplified by PCR with the primers BamHI-5’hCCR5 and HindIII-3’hCCR5. Subsequently, the PCR products were cloned into PUC19 plasmids using the BamHI and HindIII restriction sites. CCR5 disruption was confirmed by sequence analysis of individual cloned transformants.
This experiments was approved by the Human Subject Research Ethics Committee (IRB), Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences with the reference number: GIBH-IRB02-2009002.
We are grateful to Miguel A. Esteban and his group for supplying the hiPSCs. This work was financially supported by the National Science and Technology Major Project (2013ZX10001-004-002-004) and the National Natural Science Foundation of China (No. 81200398).
- Mandell JG, BarbasIII CF: Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res 2006, 34: W516–523. 10.1093/nar/gkl209PubMed CentralPubMedView ArticleGoogle Scholar
- Li T, Yang B: TAL Effector Nuclease (TALEN) Engineering. Methods Mol Biol 2013, 978: 63–72. 10.1007/978-1-62703-293-3_5PubMedView ArticleGoogle Scholar
- Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF: Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 2010, 186: 757–761. 10.1534/genetics.110.120717PubMed CentralPubMedView ArticleGoogle Scholar
- Collin J, Lako M: Concise review: putting a finger on stem cell biology: zinc finger nuclease-driven targeted genetic editing in human pluripotent stem cells. Stem Cells 2011, 29: 1021–1033. 10.1002/stem.658PubMedView ArticleGoogle Scholar
- Zou J, Maeder ML, Mali P, Pruett-Miller SM, Thibodeau-Beganny S, Chou BK, Chen G, Ye Z, Park IH, Daley GQ, Porteus MH, Joung JK, Cheng L: Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 2009, 5: 97–110. 10.1016/j.stem.2009.05.023PubMed CentralPubMedView ArticleGoogle Scholar
- Hockemeyer D, Wang HY, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng XD, Hinkley SJ, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R: Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 2011, 29: 731–734. 10.1038/nbt.1927PubMed CentralPubMedView ArticleGoogle Scholar
- Ding Q, Lee YK, Schaefer EA, Peters DT, Veres A, Kim K, Kuperwasser N, Motola DL, Meissner TB, Hendriks WT, Trevisan M, Gupta RM, Moisan A, Banks E, Friesen M, Schinzel RT, Xia F, Tang A, Xia Y, Figueroa E, Wann A, Ahfeldt T, Daheron L, Zhang F, Rubin LL, Peng LF, Chung RT, Musunuru K, Cowan CA: A TALEN Genome-Editing System for Generating Human Stem Cell-Based Disease Models. Cell Stem Cell 2013, 12: 238–251. 10.1016/j.stem.2012.11.011PubMed CentralPubMedView ArticleGoogle Scholar
- Thomas CE, Ehrhardt A, Kay MA: Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 2003, 4: 346–358. 10.1038/nrg1066PubMedView ArticleGoogle Scholar
- Morris MC, Deshayes S, Heitz F, Divita G: Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biol Cell 2008, 100: 201–217. 10.1042/BC20070116PubMedView ArticleGoogle Scholar
- Vyas PM, Payne RM: TAT opens the door. Mol Ther 2008, 16: 647–648. 10.1038/mt.2008.24PubMed CentralPubMedView ArticleGoogle Scholar
- Brasseur R, Divita G: Happy birthday cell penetrating peptides: already 20 years. Biochim Biophys Acta 2010, 1798: 2177–2181. 10.1016/j.bbamem.2010.09.001PubMedView ArticleGoogle Scholar
- Lopalco L: CCR5: From Natural Resistance to a New Anti-HIV Strategy. Viruses 2010, 2: 574–600. 10.3390/v2020574PubMed CentralPubMedView ArticleGoogle Scholar
- Hutter G, Nowak D, Mossner M, Ganepola S, Mussig A, Allers K, Schneider T, Hofmann J, Kucherer C, Blau O, Blau IW, Hofmann WK, Thiel E: Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med 2009, 360: 692–698. 10.1056/NEJMoa0802905PubMedView ArticleGoogle Scholar
- Allers K, Hutter G, Hofmann J, Loddenkemper C, Rieger K, Thiel E, Schneider T: Evidence for the cure of HIV infection by CCR5Delta32/Delta32 stem cell transplantation. Blood 2011, 117: 2791–2799. 10.1182/blood-2010-09-309591PubMedView ArticleGoogle Scholar
- Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee YL, Guschin DY, Rupniewski I, Waite AJ, Carpenito C, Carroll RG, Orange JS, Urnov FD, Rebar EJ, Ando D, Gregory PD, Riley JL, Holmes MC, June CH: Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 2008, 26: 808–816. 10.1038/nbt1410PubMed CentralPubMedView ArticleGoogle Scholar
- Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, Crooks GM, Kohn DB, Gregory PD, Holmes MC, Cannon PM: Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol 2010, 28: 839–847. 10.1038/nbt.1663PubMed CentralPubMedView ArticleGoogle Scholar
- Symonds GP, Johnstone HA, Millington ML, Boyd MP, Burke BP, Breton LR: The use of cell-delivered gene therapy for the treatment of HIV/AIDS. Immunol Res 2010, 48: 84–98. 10.1007/s12026-010-8169-7PubMedView ArticleGoogle Scholar
- Huddleston JE: HIV: HIV hides in haematopoietic stem cells. Nat Rev Microbiol 2011, 9: 311. 10.1038/nrmicro2564PubMedView ArticleGoogle Scholar
- Yao Y, Nashun B, Zhou T, Qin L, Zhao S, Xu J, Esteban MA, Chen X: Generation of CD34+ Cells from CCR5-disrupted Human Embryonic and Induced Pluripotent Stem Cells. Hum Gene Ther 2011, 23: 238–242.PubMedView ArticleGoogle Scholar
- Wang Z, Troilo PJ, Wang X, Griffiths TG, Pacchione SJ, Barnum AB, Harper LB, Pauley CJ, Niu Z, Denisova L, Follmer TT, Rizzuto G, Ciliberto G, Fattori E, Monica NL, Manam S, Ledwith BJ: Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation. Gene Ther 2004, 11: 711–721. 10.1038/sj.gt.3302213PubMedView ArticleGoogle Scholar
- Planel S, Salomon A, Jalinot P, Feige JJ, Cherradi N: A novel concept in antiangiogenic and antitumoral therapy: multitarget destabilization of short-lived mRNAs by the zinc finger protein ZFP36L1. Oncogene 2010, 29: 5989–6003. 10.1038/onc.2010.341PubMedView ArticleGoogle Scholar
- Nain V, Sahi S, Verma A: CPP-ZFN: a potential DNA-targeting anti-malarial drug. Malar J 2010, 9: 258. 10.1186/1475-2875-9-258PubMed CentralPubMedView ArticleGoogle Scholar
- Gaj T, Guo J, Kato Y, Sirk SJ, Barbas III CF: Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods 2012, 9: 805–807. 10.1038/nmeth.2030PubMed CentralPubMedView ArticleGoogle Scholar
- Chen S, Oikonomou G, Chiu CN, Niles BJ, Liu J, Lee DA, Antoshechkin I, Prober DA: A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly. Nucleic Acids Res 2013, 41: 2769–2778. 10.1093/nar/gks1356PubMed CentralPubMedView ArticleGoogle Scholar
- Liu J, Li C, Yu Z, Huang P, Wu H, Wei C, Zhu N, Shen Y, Chen Y, Zhang B, Deng WM, Jiao R: Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy. J Genet Genomics 2012, 39: 209–215. 10.1016/j.jgg.2012.04.003PubMedView ArticleGoogle Scholar
- 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: e82. 10.1093/nar/gkr218PubMed CentralPubMedView ArticleGoogle Scholar
- Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK: FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 2012, 30: 460–465. 10.1038/nbt.2170PubMed CentralPubMedView ArticleGoogle Scholar
- Doyon Y, Choi VM, Xia DF, Vo TD, Gregory PD, Holmes MC: Transient cold shock enhances zinc-finger nuclease-mediated gene disruption. Nat Methods 2010, 7: 459–460. 10.1038/nmeth.1456PubMedView ArticleGoogle Scholar
- 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: 143–148. 10.1038/nbt.1755PubMedView ArticleGoogle Scholar
- Tovkach A, Zeevi V, Tzfira T: A toolbox and procedural notes for characterizing novel zinc finger nucleases for genome editing in plant cells. Plant J 2009, 57: 747–757. 10.1111/j.1365-313X.2008.03718.xPubMedView ArticleGoogle Scholar
- Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ: A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 2010, 649: 247–256. 10.1007/978-1-60761-753-2_15PubMedView ArticleGoogle Scholar
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