Methods xxx (2014) xxx–xxx

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TALEN utilization in rice genome modifications Ting Li, Bo Liu, Chih Ying Chen, Bing Yang ⇑ Department of Genetics, Development and Cell Biology, Iowa State University, Ames, USA

a r t i c l e

i n f o

Article history: Received 17 November 2013 Revised 10 March 2014 Accepted 17 March 2014 Available online xxxx Keywords: TALEN TAL effector nuclease Rice Gene editing Genome engineering Xanthomonas

a b s t r a c t Transcription activator-like effector nucleases (TALENs), the newly developed and powerful genetic tools for precise genome editing, are fusion proteins of TAL effectors as DNA binding domains and the cleavage domain of FokI endonuclease. As a pair, the central repeat regions of TALENs determine the DNA binding specificity for the two sub-target sites; and the dimeric non-specific FokI cleavage domains cause a DNA double strand break (DSB) between the bound sequences. In vivo, cells repair the DSBs through either non-homologous end joining (NHEJ) pathway or homologous recombination (HR) pathway. Various methods have been developed for easy and fast assembly of TALEN genes for their utilization in a variety of eukaryotic cells or organisms. Here we present a TALEN-based rice genome modification protocol including constructing modularly assembled TALENs, rice transformation, and mutant screening. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Transcription activator-like effectors (TALEs) represent a large group of effector proteins that are of bacterial origin and translocated into host cells via a type III secretion system during pathogen infection. They represent the largest family of type III effectors and have been discovered almost exclusively in certain strains of bacterial plant pathogens in Xanthomonas and Ralstonia [1]. Each TALE contains a central repetitive region consisting of a variable number of 33–35 amino acid repeats that are nearly identical except for the two variable amino acids at the positions 12 and 13, known as repeat variable di-residues (RVDs). A combination of repeat number and composition of RVDs determines the specificity of each TALEN for its target DNA sequence. The recognition of DNA target by the TALE repeat domains appear to follow a simple code, i.e., one repeat corresponds to one nucleotide and one type of RVDs preferentially recognizes one type of four nucleotides of target [2,3]. Repeats with four prevalent RVDs (HD, NI, NG and NN corresponding to nucleotide C, A, T and G, respectively) have been used to assemble novel TALEs or repeat domains targeting Abbreviations: TALE, transcription activator-like effector; TALEN, transcription activator-like effector nuclease; DSB, DNA double strand break; RVD, repeat variable di-residues; EBE, effector binding element; NHEJ, non-homologous endjoining; HR, homologous recombination; SSA, single strand annealing; MCS, multiple cloning sites; SC drop-out, synthetic complete drop-out; hptII, hygromycin resistance gene; T0, primary transgenic plant. ⇑ Corresponding author. Address: 1035C Roy J. Carver Co-Lab, Iowa State University, Ames, IA 50011, USA. Fax: +1 515 294 5256. E-mail address: [email protected] (B. Yang).

the preselected DNA sequences [4–7]. The C-terminal nuclear localization signals (NLSs) and acidic activation domain (AD) are characteristic features of eukaryotic transcription factors [8,9]. And these two motifs are required for the avirulence activities of most and virulence functions of all TALEs analyzed [10] (Fig. 1A). TALE nucleases (TALENs), fusion proteins of the full-length or truncated TALEs and the DNA cleavage domain of FokI endonuclease [11,12], are newly developed and highly efficient tools for targeted gene modification. Like zinc finger nucleases, fusion proteins of zinc finger motifs and the DNA cleavage domain of FokI [13,14], TALENs also work as the dimer by binding to two adjacent effector binding elements (EBEs), which are separated by a spacer. The optimal length of the spacer is mainly determined by the C-terminal length between the TALE repeat domain and the FokI cleavage domain [6,7] (Fig. 1B). The dimeric FokI domains cause DSBs by non-specifically cutting the spacer DNA, and the subsequent DNA repair pathway is activated to seal the break through NHEJ or HR, leading to the specific DNA sequence alterations at or near the cleft site [15]. DNA repair through NHEJ often results in mutagenic deletions/insertions at the site of DNA breakage, while HR can be exploited to make a small change to an endogenous gene or insert a genetic element by providing an exogenous ‘‘donor’’ DNA fragment that is homologous to the target site sequences [16,17] (Fig. 1C). TALENs have been widely applied in yeast [7], animals [18–21] and even human pluripotent cells [22]. For plant, the technology for targeted genome modification is critical for improvement of crop yield and resistance to diverse biotic and abiotic stresses, as well as for accelerating the development of those beneficial traits

http://dx.doi.org/10.1016/j.ymeth.2014.03.019 1046-2023/Ó 2014 Elsevier Inc. All rights reserved.

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2. Materials 2.1. Molecular cloning 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12.

TALE assembly plasmid kit (as described [27]). Restriction enzymes (e.g., Fermentas). Alkaline Phosphatase, Calf Intestinal (CIP) (e.g., Promega). RNase A (e.g., Fermentas). 1 kb DNA Ladder (Invitrogen). Bacterial competent cells. Luria–Bertani (LB) broth medium with appropriate antibiotics, ampicillin (100 mg/L), kanamycin (50 mg/L), rifampicin (30 mg/L). T4 DNA ligase (e.g., New England Biolabs). GENECLEAN III Kit for DNA purification. Apparatus for DNA electrophoresis. 30 and 37 °C incubators. 30 and 37 °C shakers.

2.2. Yeast transformation and single strand annealing (SSA) assay 2.2.1. Yeast medium

Fig. 1. Genome modification with one pair of sequence-specific TALENs. (A) Schematic of TALENs used in the study. Full-length TALE is fused with the homodimeric cleavage domain of FokI (FN). The number of amino acids in the separate domains is shown above each region (NLS, nuclear localization signals; AD, transcription activation domain). (B) Schematics of the active TALEN/DNA complex. (C) Double strand break (DSB) induced by one pair of TALENs activates DNA repair mechanisms either by homologous recombination (HR) or non-homologous end joining (NHEJ). HR, stimulated by the homologous DNA template (double-stranded or single-stranded donor DNA) leads to gene replacement (left panel). Through NHEJ, DSB leads to frame-shift mutations with small insertions or deletions and results in gene disruption (right panel).

in crops. For example, TALENs have been used to modify the promoter region of the blight disease susceptibility (S) gene (OsSWEET14) in rice. The precise modifications disrupted the interaction between the virulent TALEs and the S gene promoter and, consequently, prevented the S gene from induction and infection by the pathogen Xanthomonas oryzae pv. oryzae [23]. It has also been demonstrated that TALENs are capable of creating targeted gene modifications in other monocot species, such as Brachypodium and barley [24,25]. In dicot species, TALENs can not only modify genomes in the protoplasts of tobacco and Arabidopsis, but also produce heritable mutations in Arabidopsis [26]. TALEN-based genome editing in plant involves (1) design and synthesize novel TALEN genes based on the preselected target DNA sequences, (2) construct plasmid expressing the paired TALEN genes (probably along with a ‘‘donor’’ DNA), (3) transform and regenerate transgenic plants of TALENs, (4) screen primary or progeny plants for desired modifications, and (5) obtain progeny of modified plants that contain the gene modification and removal of TALEN transgene through genetic segregation from the self- or out-crossed progeny (Fig. 5). In this article, we provide protocols for construction of TALEN-mediated genome editing system and further describe the strategy for detecting of mutation events, a strategy that was successfully applied in rice as described previously [23]. The step-by-step protocols described here for rice can be, in principle, easily adaptable to other monocots and even for dicot genome modification.

1. YPAD medium: 6.0 g yeast extract (Difco), 12.0 g peptone (Difco), 12.0 g glucose, 60 mg adenine hemisulphate, 10.0 g Bacto-agar (Difco) and 600 ml distilled water. 2. Synthetic complete drop out (SC drop-out) medium: 4.0 g Difco yeast nitrogen base (without amino acids), 12.0 g glucose, 0.5 g SC drop-out mix, 10.0 g Bacto-agar (Difco) and 600 ml distilled water, pH 5.6. 2.2.2. Yeast transformation reagents 1. 2. 3. 4.

Single-stranded carrier DNA (2 mg/ml). Lithium acetate stock solution (1.0 M). Polyethylene glycol 3350 (PEG 50% w/v). Yeast plasmid DNA.

2.2.3. Yeast SSA assay 1. Yeast b-galactosidase assay kit (Thermo Fisher Scientific, Rockford, IL, USA). 2. Absorbance plate reader (VWR). 3. Spectrophotometer (Bio-Rad). 2.3. Rice tissue culture and transformation 2.3.1. Plant expression vectors and Agrobacterium tumefaciens strain 1. pCAMBIA1300 (Cambia). 2. pEH3 (available upon request). 3. A. tumefaciens strain EHA105. 2.3.2. Rice cultivar The immature embryos of the Japonica rice (Oryza sativa L.) cultivar Nipponbare or Kitaake were used in this study. 2.3.3. Reagents and medium for tissue culture and transformation 1. Callus initiation and induction. MSD medium: 4.4 g/L Murashige and Skoog (MS) basal medium with Gamborg vitamins (PhytoTechnology Laboratories), 30 g/L sucrose, 2 mg/L 2,4-dichlorophenoxyacetic acid, and 8 g/L agar (PhytoTechnology Laboratories), pH 5.8.

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2. Medium for A. tumefaciens strain EHA105. TY medium: 5.0 g/L tryptone, 3.0 g/L yeast extract, 50 mg/L kanamycin, 30 mg/L rifampicin, or 15 g/L agar for solid medium, pH 5.5. 3. Infection of calli with Agrobacterium. MSD + AS medium: 4.4 g/L Murashige and Skoog (MS) basal medium with Gamborg vitamins (PhytoTechnology Laboratories), 30 g/L sucrose, 2 mg/L 2,4-dichlorophenoxyacetic acid, 0.2 mM acetosyringone (AS) (Sigma–Aldrich), or 8 g/L agar for solid medium (PhytoTechnology Laboratories), pH 5.8. 4. Selection of transgenic calli. 4.4 g/L Murashige and Skoog (MS) basal medium with Gamborg vitamins (PhytoTechnology Laboratories), 30 g/L sucrose, 2 mg/L 2,4-dichlorophenoxyacetic acid, 400 mg/L Timentin (GlaxoSmithKline), 50 mg/L hygromycin B (Sigma–Aldrich), and 8 g/L agar (PhytoTechnology Laboratories), pH 5.8. 5. Regeneration of plants. 4.4 g/L Murashige and Skoog (MS) basal medium with Gamborg vitamins (PhytoTechnology Laboratories), 30 g/L Sucrose, 50 g/L sorbitol, 3 mg/L 6-benzylaminopurine (BAP) (Sigma–Aldrich), 0.5 mg/L 1-naphthaleneacetic acid (NAA) (Sigma–Aldrich), 50 mg/L hygromycin B (Sigma–Aldrich), and 12 g/L agar (PhytoTechnology Laboratories), pH 5.8. 6. Root induction. 1/2 MS medium: 2.2 g/L Murashige and Skoog (MS) basal medium with Gamborg vitamins (PhytoTechnology Laboratories), 10 g/L sucrose, 50 mg/L hygromycin B (Sigma– Aldrich), 6 g/L agargellan (PhytoTechnology Laboratories), pH 5.8. 2.4. Analysis of transgenic rice callus lines or plants 2.4.1. Plant genomic DNA extraction 1. Suspension buffer (pH 8): 50 mM EDTA, 120 mM Tris–HCl, 1 M NaCl, 0.5 M sucrose, 2% Triton-X 100, and 0.2% b-mercaptoethanol (to be freshly added just before use). 2. PVP (Polyvinyl pyrrolidone, MW 10,000, Sigma). 3. Mortar and pestle. 4. Extraction buffer: 20 mM EDTA, 100 mM Tris–HCl, 1.5 M NaCl, 2% CTAB, 1% b-mercaptoethanol. 5. Water bath. 6. Chloroform:isoamyl alcohol (24:1). 7. Isopropanol. 8. 70% Ethanol. 9. RNase A. 10. TE buffer: 10 mM Tris–HCl, 1 mM EDTA, pH = 8.0. 2.4.2. Genotyping by PCR 1. T7 endonuclease I (T7E1) (New England Biolabs). 2. Restriction enzymes.

3. 4. 5. 6.

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Taq DNA polymerase. Deoxynucleotide mixture (10 mM) (e.g., New England Biolabs). Primers. PCR thermal cycler (Bio-Rad).

3. Methods 3.1. Design and engineer TALENs through Golden Gate cloning We usually design and engineer TALEN genes encoding 16–24 repeats using a protocol described in a book chapter [27]. Briefly, two steps are used for the modular assembly method. Golden Gate cloning strategy was adopted to assemble two (if fewer than 16 repeats in the final TALEN genes) or three repeat fragment (if more than 16) each in an intermediate plasmid. Then the two or three fragments are released with restriction enzyme and put together into TALEN-scaffold to obtain plasmid pSK-TALEN-L or pSKTALEN-R. The full length and AD-truncated backbones of TAL effector gene avrXa7 are fused with the plant codon optimized FokI cleavage domain in this study. Longer repeat domain of TALEN may have higher specificity and less possibility of off-target effect. However, it may compromise abundance of TALEN proteins expressed in cells. Lower level of protein expression appears to result in lower efficiency of gene modification (data not shown). Furthermore, the rice codon optimized TALEN genes with N- and C-terminal truncations (e.g., a deletion of N-terminal 153 amino acids and presence of C-terminus with only 63 amino acids as described by Miller et al. [6] fused with hetero-dimeric FokI cleavage domains may worth to explore, which may achieve the higher gene-modification efficiency and reduce off-target effect. To select the two sub-target sites (or effector binding elements, EBEs) for the paired TALENs, we use the following criteria, (i) the 50 ‘‘T’’ preceding each target sequence; (ii) a 18–20 bp spacer between the two EBEs, and (iii) within the spacer region, usually in the middle of the spacer, a restriction site is preferred. In this case, both T7E1 and restriction enzyme can be used to screen for potential site-specific modifications. Fig. 2 shows an example for one of the TALEN target sites in Os11N3 (or OsSWEET14) promoter region. The plasmid library of the TALEN assembly kit used in this study is generated based on Golden Gate cloning strategy. The kit is available upon request. 3.2. Yeast homologous recombination based assay as a quick test for TALEN activity Before transforming the TALEN constructs into plants, it is important to make sure that encoded TALENs are active and specific. In yeast cells, transient co-expression of TALENs and reporter genes, such as LacZ, which encodes b-galactosidase, and fused with the TALEN EBE sites is a simple and effective way to test the TALEN activities in short period. Homologous recombination (or single strand annealing) based repair to the TALEN-induced DSB could reconstitute the two halves of nonfunctional reporter gene that

Fig. 2. Schematics of TALEN DNA target site in the Os11N3 promoter of the rice chromosome 11. TALEN-L and TALEN-R (red arrow) recognize and bind to the EBE sites (underlined in black) beginning with T0 (in red). The two EBEs are separated by a spacer (lowercase letters) that contains BstNI recognition site (in green). PCR amplifies 400 bp promoter region centered the target site with primers FP and RP.

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Fig. 3. Schematic of yeast-based TALEN activity assay. TALEN expression plasmids are co-transformed into yeast strain YPH499. The reporter plasmid that contains the TALEN target site is introduced into yeast strain YPH500. After combining the three plasmids together through mating the two strains, TALENs bind and cleave within the target sites. This assay measures TALEN ability to stimulate the plasmidborne homologous recombination between duplicated regions of lacZ gene, resulting in a functional LacZ gene.

are separated by the sequence of EBE sites into a functional version [12] (Fig. 3). 3.2.1. Make yeast expression and reporter constructs 1. Digest the empty vector pCP3M with the restriction enzymes BamHI and SpeI (1 ll of each restriction enzyme with 1 lg of DNA for 2 h at 37 °C) to create vector DNA. Digest pSK-TALENL plasmid (product from step 3.1) with BglII, SpeI and ScaI (1 ll of each restriction enzyme with 2 lg of DNA for 2 h at 37 °C) to release the insert DNA. Separate the vector and insert DNA through electrophoresis and purify the DNA using the GENECLEAN III Kit. Ligation reaction is set with T4 DNA ligase for 2 h at room temperature. Transform the ligation product into the XL1-Blue competent cells, screen the correct clones for further yeast transformation. The TALEN-L gene is under the control of the yeast TEF1 promoter and CYC1 terminator. Note 1: BglII and BamHI generate compatible 50 overhang ends for ligation. Note 2: The purpose of using ScaI is to cut the pBluescript-SK backbone into two smaller pieces (1.8 and 1.2 kb) for better separation of the TALEN-L coding region from the backbone fragment in pSK-TALEN-L to prevent the potential problem that the insert ligates back to the contaminated backbone forming

pSK-TALEN-L again, which would interfere the screen of the correct clones because both the pCP3M and pSK plasmids are ampicillin resistant. 2. Similarly, another TALEN gene can be constructed into pCP4M from pSK-TALEN-R. Digest pCP4M with BamHI and SpeI, and pSK-TALEN-R with BglII, SpeI and ScaI to release TALEN-R gene coding region, and ligate them together to generate pCP4TALEN-R for yeast transformation. 3. Construct the pCP5-EBE reporter plasmid. Digest pCP5 vector with BglII and SpeI, then separate and purify the 11.3 kb backbone from the 2.5 kb fragment, which contains the expression cassette of ccdB and chloramphenicol acetyltransferase (CAT) gene. Design and synthesize a pair of oligonucleotides (oligoF, oligoR) that, after annealing, contain a TALEN target EBE site and two overhang ends that are compatible with the respective ends created by BglII and SpeI in pCP5 (Table 1). Mix 50 fmol of each of these oligonucleotides together in 100 ll of 1 Taq DNA polymerase buffer, heat the solution in boiling water for 5 min and then allowing the mixture to cool down slowly to room temperature. Finally, ligate annealed oligonucleotides with the pCP5 vector DNA, and identify the correct clones. Note 3: pCP5 is a kanamycin resistant and low copy plasmid; the kanamycin working concentration for this low copy plasmid is 15 mg/L. Note 4: Since ccdB is a lethal gene for positive selection by killing the background cells with the empty vector, most clones picked up from the grown colonies should contain plasmids containing insert. Note 5: pCP5-EBE plasmid contains the targeted EBE site, which is flanked by a 125 bp duplicated region of lacZ gene that recombines together to extrude the disrupted fragment to form a functional lacZ gene if the paired TALENs are active to cause DSBs in yeast cells. 3.2.2. Test TALEN nuclease activity in yeast SSA assay 1. Transform both pCP3-TALEN-L and pCP4-TALEN-R plasmids into yeast mating strain YPH499 (MATa ura3-52 lys2801_amber ade2-101_ochre trp1-D63 his3-D200 leu2-D1). A combination of pCP3M and pCP4M empty vectors and a combination of pCP3-AvrXa7-FokI and pCP4-PthXo1-FokI plasmids are used as the negative and positive control, respectively. Plate the transformed cells on SC drop-out medium lacking histidine (for pCP3M and its derivatives) and leucine (for pCP4M and its derivatives). Then transform the reporter plasmid pCP5-EBE containing the target site for the tested TALENs into the yeast mating strain YPH500 (MATa ura3-52 lys2-801_amber ade2101_ochre trp1-D63 his3-D200 leu2-D1). The plasmid pCP5– 8/11N3 containing the EBEs of AvrXa7 and PthXo1 is used as a positive control. Plate the transformed cells on SC drop-out medium lacking tryptophan. Yeast transformation is performed with a high efficiency transformation protocol developed by Gietz lab [28].

Table 1 Primer sequences. Primers

Sequences

FP RP OligoF OligoR P1F P1R P2F P2R

50 -CATGGCTGTGATTGATCAGG-30 50 -CCGGATCCAGCCATTGCAGCAAGATCTTG-30 50 -GATCTCTTCCTTCCTAGCACTATATAAACCCCCTCCAACCAGGTGCTAAGCTCATCAAGCCTTCAAGCA-30 50 -CTAGTGCTTGAAGGCTTGATGAGCTTAGCACCTGGTTGGAGGGGGTTTATATAGTGCTAGGAAGGAAGA-30 50 -CCGCTCGTCTGGCTAAGATC-30 50 -CGCTGAAATCACCAGTCTCTC-30 50 -CAGCTAGTGAAATCTGAATTGG-30 50 -CATCGCAAGACCGGCAACAGG-30

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2. About 2 days post transformation at 30 °C incubator, pick single colony in triplicate from each transformant and grow them in histidine and leucine minus SC drop-out liquid medium (for TALEN expressing yeast) and tryptophan free SC drop-out liquid medium (for reporter yeast) overnight. 3. Next day, mix the same number of yeast cells (1  106) from YPH499 containing the paired TALEN plasmids and YPH500 containing pCP5-EBE plasmids together; also mix YPH499 containing pCP3M and pCP4M empty vectors and YPH500 containing pCP5-EBE together as a negative control and mix YPH499 containing pCP3-AvrXa7-FokI and pCP4-PthXo1-FokI and YPH500 containing pCP5–8/11N3 as a positive control in 500 ll YPAD medium and grow the yeast cells at 30 °C for at least 6 h. 4. Wash the mated cells twice with SC drop-out liquid medium lacking histidine, leucine and tryptophan, then culture the cells in the same medium (5 ml) at 30 °C with shaking overnight to select for the mated cells and allow b-galactosidase to be expressed and accumulate. 5. Take the same amount of yeast cells for quantitative measurement of b-galactosidase activity by using the yeast b-galactosidase assay kit following the manufacture’s manual. Enzyme activity is calculated based on the equation: b-galactosidase activity = (1000  A420)/(t  V  OD660), t = time of incubation in minutes, V = volume of cells used in the assay in ml. Note 6: A yeast suspension with 0.1 of OD600 usually contains 1  106 cells/ml. 3.3. Generate TALENs transgenic rice Currently there are two popular rice transformation methods – particle bombardment and Agrobacterium-mediated transformation. Usually Agrobacterium-mediated transformation is expected to produce transformants carrying 1–1.5 copies on average of the transgene. The transformation efficiency obtained by Agrobacterium-mediated gene transfer is about 10–30%, which is lower than particle bombardment methods. Here we present the Agrobacterium-mediated rice transformation with TALEN constructs through using calli derived from the scutella of immature seeds. The transformation protocol is modified from the method described by Hiei et al. [29]. 3.3.1. Construct TALEN-expression plasmid Once confirmed for high activity in yeast SSA assay, the paired TALEN genes are inserted into the rice gene expression vectors. Because of the limited transformation efficiency, it is hard to transfer two individual T-DNAs simultaneously into the same rice callus cells through Agrobacterium-mediated gene transfer. Therefore we combine the pair (both left and right) of TALEN expression cassettes in a single T-DNA, though the T-DNA size becomes relatively large (around 15.7 kb). 1. The pCAMBIA1300-based binary vector p35S contains an expression cassette of the CaMV 35S promoter, multiple cloning sites (MCS) and Nos terminator (unpublished). The orientation of this cassette is the same as that of the CaMV 35S promoter and the CaMV 35S polyA for the expression of the hygromycin B resistance gene (hptII). Digest p35S with BamHI and SpeI (1 lg of plasmid DNA with 1 ll of each restriction enzyme, 2 h at 37 °C) for vector DNA. Use the same method to obtain DNA fragment from pSK-TALEN-L with restriction of BglII, SpeI and ScaI as to prepare the DNA fragment for yeast expression construct. The resultant binary vector contains the TALEN-L gene under the CaMV 35S promoter and fused with Nos terminator.

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2. To construct the TALEN-R expression cassette, the plasmid pEH3, a pUC19-based vector containing the maize ubiquitin 1 (Ubi1) promoter [30], MCS and Nos terminator, is digested with BamHI and SpeI (1 lg of plasmid DNA with 1 ll of each restriction enzyme, 2 h at 37 °C) to obtain vector DNA. The same method is used to obtain the TALEN-R gene fragment from pSK-TALEN-R with BglII, SpeI and ScaI as for yeast expression constructs. Ligate the insert and vector DNA and screen for the correct clones. The resultant expression cassette contains the TALEN-R under the control of the Ubi1 promoter and the Nos terminator. Note 7: pEH3 contains two HindIII recognition sites flanking the expression cassette. HindIII restriction digestion releases the expression cassette from the backbone (2.7 kb). 3. To combine the two TALEN-L and TALEN-R expression cassettes into a single binary vector, the HindIII-released TALEN-R cassette is ligated into the HindIII-cut and phosphatase-treated p35S-TALEN-L. The HindIII digestion of pEH3-TALEN-R releases an expression cassette of about 6.9 kb (2.1 kb of Ubi1 and 0.3 kb of terminator plus the size of TALEN-R). And the Calf Intestinal Phosphatase (CIP) treatment (1 unit at 37 °C for 30 min) following the HindIII digestion of p35S-TALEN-L prevents the vector from self-ligation. The subcloning results in a single binary plasmid p1300-TALEN-LR expressing the paired TALENs and the selection marker gene (htpII) in plant cells once transformed (Fig. 4). Note 8: Since the TALEN-R cassette is inserted into p35S-TALENL at a single HindIII restriction site, there are two possible orientations of insertion, i.e., TALEN-L and -R in the same or opposite direction of their respective promoter (Fig. 4A, B). The construct with a configuration of the same direction usually produces more mutation lines probably due to higher expression of TALEN genes. Note 9: To highly express TALEN-L and TALEN-R separately, we choose the CaMV 35S promoter that activates strong expression in both dicot and monocot plants and Ubi1 promoter that works well only in monocot plant (data not shown). In order to construct TALEN expression plasmid suitable for gene editing in dicot plants, the Ubi1 promoter should be replaced with another dicot-effective promoter in a combination with the 35S promoter. If two 35S promoters were used for TALEN-L and TALEN-R, the expression of both TALEN genes are silenced when tested in a transient expression system with Nicotiana benthamiana leaves through Agrobacterium-mediated gene transfer (data not shown). Alternatively, two TALEN genes can be expressed under one strong promoter (e.g., 35S promoter) if they are linked by a 2A-peptide sequence. 3.3.2. Transfer TALEN construct into rice callus cells for gene editing 1. Transform the Agrobacterium strain EHA105 with the rice expression plasmid by electroporation. Note 10: The binary vector pCAMBIA1300 is kanamycin resistant, so it is not suitable to use A. tumefaciens strains (e.g., EHA101) that are kanamycin resistant. 2. Callus initiation: Sterilize the dehusked immature seeds with 50% bleach (3% sodium hypochlorite) for 20 min, and rinse three times with sterilized water. Then place the seeds on MSD medium and incubate at 30 °C with continuous light for 14 days to initiate actively growing calli (Fig. 5A). 3. Agrobacterium infection: Grow TALEN containing Agrobacterium culture to 1.0–2.0 of OD600, mix 2 ml of Agrobacterium cells with 20 ml of liquid MSD medium (with 0.2 mM acetosyringone, pH 5.2), and immerse the rice calli in the suspension for

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Fig. 4. Schematic diagram of a single binary vector with two gene expression cassettes designed for Agrobacterium-mediated rice transformation. The expression cassette includes a promoter [maize ubiquitin 1 promoter (Ubi1) (purple box) to drive expression of the TALEN-R gene (red arrow), the cauliflower mosaic virus 35S gene promoter (35S) (green box) to allow transcription of the TALEN-L gene (red arrow)] and a gene terminator (Nos-T) (black box). The plant transformation selection marker hptII gene (light blue arrow) is under control of 35S (green box). KanR denotes the kanamycin resistant marker for bacterial selection. TALEN-L and -R coding genes are inserted between the promoter and terminator by BglII/BamHI and SpeI (BglII is in the 50 TALEN coding region, BamHI is in the MCS linking the promoter and terminator). The whole TALEN-R expression cassette is cloned into the vector by single restriction site (HindIII) after the TALEN-L expression cassette is cloned into the binary vector. LB, Left Border; RB, Right Border. (A) TALEN-L and TALEN-R is in the same direction. (B) TALEN-L and TALEN-R is in the opposite direction.

4.

5.

6. 7.

30 min. Blot dry the calli with filter paper, transfer the calli to MSD + AS plate and keep the calli in the dark at room temperature for 2–3 days. Transformed calli selection: Transfer the infected calli to selection medium (MSD supplemented with 50 mg/L of hygromycin B and 400 mg/L of Timentin) at an interval of 2 weeks for transformed callus line selection (Fig. 5B). The tissue culture and regeneration at this and subsequent stages are performed in growth chamber at 30 °C and under continuous light. Plantlet regeneration: Transfer the individual hygromycin resistant callus lines to regeneration medium to regenerate transgenic plants (Fig. 5C). Root induction: Transfer the regenerated plants into ½ MS medium for root induction (Fig. 5D). Transfer the plantlets into soil and grow them in the growth chamber with the temperature of 28 °C, relative humidity of 75%, and photoperiod of 12 h till plants are mature and seeds are harvested.

3.4. Screen and identify TALEN-modified plants 3.4.1. Extract genomic DNA from transformed calli or plants After obtain the transgenic calli, for each line, use a portion of calli to extract genomic DNA using the CTAP method [31], save others for plant regeneration (Fig. 5C). Similarly, the leaf tissues of the primary transgenic (T0) plants can be used to extract genomic DNA. PCR amplify the relevant region (500 bp) centered on the TALEN target site with specific primers and the genomic DNA as template (Fig. 2). 3.4.2. Screen for modified sequences through digestion with restriction enzymes Sometimes if the restriction sites exist within the spacer region of TALEN target site, the restriction digestion of PCR products can be used to detect the presence of modifications. For example, a BstNI restriction site is present in the middle of the spacer region of OsSWEET14, and the correspondent restriction enzyme BstNI is useful to initially screen the transformed calli or plants for putative modifications (Fig. 2). 1. Precipitate the PCR product with ethanol and dissolve the DNA in 15 ll of water.

Fig. 5. Rice transformation for TALEN-based genome editing. (A) Callus initiation. Dehusked immature kitaake seeds are cultured on the callus induction medium. Two weeks later, the proliferated calli derived from the scutella are used for Agrobacterium infection. (B) Hygromycin resistant calli grow on selection medium. (C) Shoot induction on regeneration medium. (D) Root induction for transgenic plantlets.

2. Use 5 ll of the DNA to perform the restriction digestion for 3 h. 3. Three patterns of DNA bands would be expected when the enzyme treated PCR products were fractioned in 2% agarose gel. (i) Only one band appears in the gel with the size of PCR product, suggesting that the restriction sites in both alleles are disrupted, and the lines are the candidates of bi-allelic mutations (Fig. 6A, L3, L4, L6). (ii) Three bands (uncut PCR

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product and two digested products) appear in the gel (Fig. 6A, L5), suggesting the PCR product is a mixture of DNA with the intact restriction site and destroyed restriction site. And the density of DNA bands should reflect the relative amount of PCR product with disrupted restriction site. If the uncut DNA represents about half of PCR products, it is likely that the line (callus or plant) is a mono-allelic (one of two alleles) mutant and the TALEN-induced modification occurs at one cell stage. If the density of the uncut DNA band represents only a small portion of PCR product (as indicated with two very strong smaller bands in the gel), the callus line only contains a small fraction of mutated cells or the plant contains more likely the somatic mutations. (iii) If the majority or all of the PCR products are cut (Fig. 6A, L1, L2), the spacer sequence is intact and the chance of obtain TALEN-induced modification is low. Usually the NHEJ derived mutations happen in the middle of spacer region. Therefore most of the target region would be wild type sequence if the restriction site were intact.

3.4.3. Screen for modified sequences through digestion with T7E1 An alternative way to screen callus or plant lines for mutations is using T7E1 that recognizes and cleaves double-stranded DNA at

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the mismatched site. Denature (95 °C for 5 min) and re-anneal (ramp down to 25 °C at 5 °C/min) the PCR products would make a portion of double-stranded DNA, which one strand is from the wild type and another, if any, from the mutant. Two patterns of DNA bands would be expected from T7E1 treatment when the DNA is fractionated in 2% agarose gel. (i) There is only one band (Fig. 6B, L1 and L2), suggesting no presence of mismatched site in the newly formed DNA and most likely the wild type derived PCR product. (ii) There are three bands (two smaller bands of the expected sizes and the original PCR product) (Fig. 6B, L3, L4, L5 and L6). This indicates the existence of mismatch and potential mutations other than wild type sequence within the spacer region.

3.4.4. Sequencing the PCR products of candidate lines to confirm the mutation genotypes The most convincing evidence for TALEN-induced modification must come from the sequencing data. Sequence the PCR products directly with either forward or reverse primer (Table 1) from the candidate lines after the initial screening through either restriction enzyme or T7E1 treatment. Carefully examine the sequencing chromatograms of PCR products and determine the exact pattern that may be mono-allelic (Fig. 6C, L5) or bi-allelic mutations

Fig. 6. Analysis of TALEN-based gene modification. (A) Gel picture of restriction enzyme treated PCR product (about 400 bp) amplified from the Os11N3 promoter with genespecific primers (FP & RP). A BstNI recognition site is located in the middle of spacer region and its sequence may change with the repair of TALEN-induced DSBs nearby. Three bands (about 60, 100 and 240 bp) are expected if the two alleles are wild type (lane 1 and 2), since a second BstNI recognition site is located 60 bp downstream of the 50 end. A pattern of two bands (about 340 and 60 bp) suggests a bi-allelic mutation (Lane 3, 4, 6). Four bands (about 340, 240, 100 and 60 bp) are observed if the mono-allelic mutation occurs (Lane 5). (B) Gel picture of the T7 endonuclease I treated PCR product. One band suggests no mismatch exists between the two alleles (such as lane 1 and 2). A pattern of three bands (about 160, 240 and 400 bp) indicates the mono-allelic or bi-allelic mutations (lane 3–6). (C) DNA sequencing chromatograms of three DNA fragments representing wild type (L1), bi-allelic mutation (L4) and mono-allelic mutation (L5). Sample L4 contains a 9 bp deletion in one allele and a 3 bp deletion in the other allele of Os11N3. L5 contains a wild type allele of Os11N3 and the other allele with a 33 bp deletion and a 16 bp insertion.

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(Fig. 6C, L4). Furthermore, the strength of the overlapped sequencing traces could also be an indicator of relative proportion between different genotypes. Regenerate plants from the modified callus lines or keep the mutated plants.

targeted locus modification. This sequential modification strategy is obvious useful for physiological or pathological analysis of homologous genes with functional redundancy. Acknowledgments

3.4.5. Identify the heritable mutations from progeny Harvest the seeds from the T0 plants and break the seeds dormancy through 42 °C incubation for 3 days. Grow the T1 progeny and determine the zygosity of individual plants through restriction enzyme or T7E1 digestion and sequencing confirmation. For T7E1 assay, plants with heterozygous mutations can be determined by denaturing and re-annealing their PCR products solely, followed by T7E1 treatment. The rest would be either homozygous mutants or wild type plants. Mix, denature and re-anneal them individually with wild type ones, then digest with T7E1 to differentiate the homozygous mutants. The real homozygous mutant and wild type PCR mixture would be cut by T7E1. And the identified homozygous mutant lines are ready for further phenotype analysis. Note 11: The majority of T0 plants could transmit the same genotype of mutated loci into next generation. However, some of T1 plants have larger deletions compared to the T0 plants probably due to continuous action of TALENs if the target sites are still vulnerable to TALEN binding and DSB induction. Meanwhile, mutations in T0 plants sometimes cannot be transmitted into next generation probably because the detected mutations in T0 only occur in the somatic cells. 3.4.6. Segregate out the T-DNA from progeny Self- or out-cross the T0 plants would genetically segregate out the T-DNA of selection marker and TALEN genes from the progeny plants. To determine whether the T-DNA is present in T1 plants, PCR-based assay targeting multiple locations of the T-DNA can be adopted. Genomic DNA from individual plants is used as template for PCR with pairs of primers specific for hptII (P1F & P1R) and the TALEN genes (P2F & P2R) (Table 1). Positive (p1300-TALEN-LR plasmid DNA) and negative (genomic DNA from parent line) controls should be included in the PCR-based assay. Failure in amplifying any product from the segregant indicates a T-DNA free genotype. More conclusive evidence for a T-DNA free genotype of segregant would come from the whole genome sequencing by the most advanced sequencing technologies such as Illumina or PacBio system. 3.4.7. Perform the second round of TALEN-based gene editing The seeds, harvested from T1 plants with homozygous mutation and T-DNA segregated out, are able to produce calli to perform a second round transformation with another pair of TALENs for other

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