Designing and Testing the Activities of TAL Effector Nucleases

  • Yanni Lin
  • Thomas J. Cradick
  • Gang Bao
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1114)

Abstract

Transcription activator-like effector nucleases (TALENs) have rapidly developed into a powerful tool for genome editing. To avoid labor-intensive and time-consuming experimental screening for active TALENs, a scoring system can help select optimal target sites. Here we describe a procedure to design active TALENs using a scoring system named Scoring Algorithm for Predicted TALEN Activity (SAPTA) and a method to test the activity of individual and pairs of TALENs.

Key words

Transcription activator-like effector nuclease TALEN Scoring algorithm for predicted TALEN activity (SAPTA) Gene modification Genome editing Gene targeting Gene therapy Scoring function Single-strand annealing (SSA) assay T7 endonuclease I (T7E1) mutation detection assay 

1 Introduction

Transcription activator-like (TAL) effectors are a family of DNA binding proteins, discovered in the plant pathogen Xanthomonas [1, 2, 3]. Each DNA-binding domain contains a variable number of 33–35 amino-acid repeats that specify the DNA-binding sequence primarily through their 12th and 13th repeat-variable di-residues (RVDs) [4]. Each RVD specifies one nucleotide with minimal context dependence [1, 3, 5]. TALENs target specific DNA sequences through a series of RVD-containing repeats, flanked by modified N- and C-termini [6, 7] that are linked to a FokI nuclease domain [8, 9, 10]. When a pair of TALENs binds to their specific half-sites with the correct orientation and spacing to allow the nuclease domains to dimerize, the intervening sequence is cleaved. TALENs have been used to edit genomic DNA sequences in a variety of biological systems, including human cells, rats, zebrafish, nematodes, and plants [5, 6, 7, 11, 12, 13, 14, 15].

TALENs are easy to design due to the modular nature of the DNA-binding domain. However, TALENs targeting different DNA sequences have cleavage activities varying over a wide range—sometimes the activity is below detection. Current design guidelines provide qualitative criteria for selecting TALEN target sites, but could not guarantee an efficient gene modification rate. In this chapter, we describe the use of a quantitative scoring system, SAPTA, to predict TALEN activities and select optimal TALEN target sites for robust gene modification [16]. Although SAPTA was optimized to design TALENs constructed using the NK RVD to target the nucleotide guanine, we found that it also yields highly active TALENs constructed using the NN RVD that also target guanine. The SAPTA scoring system was programmed into an online design tool to enable researchers to modify genes more effectively (http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html). We also describe assays that can be used to quantify the activities of TALENs individually, or in pairs. This assay may be important for quality control of TALEN constructs and for choosing optimal TALEN sets to study further. When there are several overlapping TALEN target sites, this assay can also be used to pick the optimal left and right monomers to test further.

2 Materials

2.1 Assembly of Homodimeric or Heterodimeric Target Plasmids

  1. 1.

    Backbone plasmids (seeNote1).

     
  2. 2.

    Oligonucleotides containing target sequences and spacers.

     
  3. 3.

    10× T4 DNA ligase reaction buffer (New England Biolabs).

     
  4. 4.

    T4 polynucleotide kinase (New England biolabs).

     
  5. 5.

    Two restriction enzymes unique to the backbone plasmid, e.g., AscI and SbfI.

     
  6. 6.

    Alkaline Phosphatase, Calf Intestinal (CIP) (New England Biolabs).

     
  7. 7.

    T4 DNA ligase (400,000 units/ml) (New England Biolabs).

     
  8. 8.

    Thermal cycler.

     
  9. 9.

    Agarose.

     
  10. 10.

    10 mg/ml ethidium bromide solution.

     
  11. 11.

    1× TAE buffer.

     
  12. 12.

    Microwave.

     
  13. 13.

    Agarose gel electrophoresis apparatus.

     
  14. 14.

    UV imaging station.

     
  15. 15.

    Gel extraction kit.

     
  16. 16.

    Tabletop microcentrifuge.

     
  17. 17.

    37 °C oven.

     
  18. 18.

    Chemically competent E. coli.

     
  19. 19.

    LB plates containing appropriate antibiotic.

     
  20. 20.

    LB broth containing appropriate antibiotic.

     
  21. 21.

    Oligonucleotide primers for colony screen.

     
  22. 22.

    GoTaq Green Master Mix (Promega).

     
  23. 23.

    Plasmid miniprep kit.

     

2.2 Cell Transfection

  1. 1.

    293T cells (ATCC, Manassas, VA).

     
  2. 2.

    293T cell culture media: Dulbecco’s Modified Eagle Medium (Sigma-Aldrich, D6429) is supplemented with 2 mM l-glutamine and 10 % FBS.

     
  3. 3.

    Any 24-well multiwell plate, sterile, tissue-culture treated, flat bottom with lid.

     
  4. 4.

    0.1 % Gelatin.

     
  5. 5.

    Control plasmid for gauging transfection efficiency, e.g., EGFP plasmid.

     
  6. 6.

    Flow cytometer (e.g., BD Accuri C6).

     
  7. 7.

    Fugene HD Transfection Reagent (Promega). Store at 4 °C with cap closed tightly.

     
  8. 8.

    Filter sterilized 2 M CaCl2. Store at room temperature.

     
  9. 9.

    Filter sterilized 2× HBS buffer: To make 100 ml buffer, dissolve 1.6 g NaCl, 1.2 g HEPES, 100 μl 1.5 M Na2HPO4 stock solution in 80 ml water. Adjust pH to 7.05 and bring the volume to 100 ml. Adjust the pH again to assure accurate pH. Make aliquots of the buffer and store at −20 °C. Once thawed, the buffer can stay stable at room temperature for several months. Do not refreeze the buffer.

     

2.3 Single Strand Annealing (SSA) Assay Using PCR

  1. 1.

    QuickExtract DNA extraction solution (Epicentre).

     
  2. 2.

    GoTaq Green Master Mix (Promega).

     
  3. 3.

    PCR tube strips with caps.

     
  4. 4.

    Oligonucleotide primers (seeNote2).

     
  5. 5.

    Vortex mixer.

     
  6. 6.

    Thermal cycler.

     
  7. 7.

    Agarose gel electrophoresis apparatus and reagents as in Subheading 2.1.

     

2.4 T7 Endonuclease I (T7E1) Mutation Detection Assay

  1. 1.

    Oligonucleotide primers (seeNote3).

     
  2. 2.

    QuickExtract DNA extraction solution (Epicentre).

     
  3. 3.

    AccuPrime Taq DNA polymerase high fidelity (Life Technologies, 12346-086 or 12346-094).

     
  4. 4.

    DMSO.

     
  5. 5.

    PCR tube strips with caps or 96-well PCR plates.

     
  6. 6.

    Thermal cycler.

     
  7. 7.

    Any PCR purification kit or gel extraction kit (seeNote4).

     
  8. 8.

    Agarose gel electrophoresis apparatus and reagents as in Subheading 2.1.

     
  9. 9.

    T7 Endonuclease I (New England Biolabs).

     
  10. 10.

    6× xylene cyanol loading dye: 4 g of sucrose, 3 mg of xylene cyanol FF (Sigma-Aldrich), add sterile water to 10 ml. Store at 4 °C.

     
  11. 11.

    0.5 M EDTA.

     

3 Methods

3.1 Design of TALENs Targeted to Genes of Interest

  1. 1.

    Obtain the genomic DNA sequence to be targeted and cleaved by TALENs. For example, if the disruption of a gene is desired, DNA sequences of the first several exons may be selected. To correct a specific mutation within a gene using the homologous-directed recombination (HDR) pathway, the sequence around the mutated site may be selected.

     
  2. 2.

    Direct an Internet browser to the SAPTA Home Page (http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html). Some instructions for using this tool are provided on the page, with links to further instructions and tutorials.

     
  3. 3.

    Paste the DNA sequence of interest into the input box below “Enter a DNA sequence” (seeNote5), and press “Submit.” Typically a sequence range from 100 to 1,500 bp may be entered. Sequence shorter than 100 bp can be searched but may not yield high scoring TALEN pairs (seeNote6). To search a sequence longer than 1,500 bp, check “Override the 1,500 base pair limit?” below the sequence input box. The Web site also allows a variety of custom settings, such as specifying the lengths of spacer and TAL repeat array for the TALEN designs. Explanations of these custom settings are found in the online tutorials (click the “Tutorials” tab located on the top of the SAPTA Web page).

     
  4. 4.

    The output from SAPTA contains a table of TALEN pairs ranked by their composite scores. The table can be copied and pasted into Microsoft Excel to save the data. The columns of this table are explained as follows.

    Starting index: the position of the 5′ end of the left TALEN target sequence. If brackets where used on the DNA sequence entered, the numbering is relative to the bracketed nucleotides (seeNote5), otherwise the numbering is relative to the first nucleotide in the searched sequence.

    Left and right TALEN sequences: the target sequences of left and right TALENs, respectively. The 5′ nucleotide is followed by a hyphen and the sequence targeted by the RVDs (e.g., T-TACTGAAGTAACCT).

    Left TALEN size, spacer size, and right TALEN size: the nucleotide lengths (bp) of each target DNA sequences.

    Left and right TALEN scores: the scores that predict the individual activities of left and right TALEN monomers, respectively. The score is calculated by the SAPTA function that was optimized based on a training set of intracellular TALEN activity data.

    Composite score: predict the activity of a pair of TALENs by functionally combining the individual scores of left and right TALENs of each pair.

    Restriction enzyme name and sequence: if there is a unique restriction enzyme site in the spacer of a TALEN pair, it will show in this column together with the sequence of this enzyme site. An enzyme site can be used for Restriction Fragment Length Polymorphism (RFLP) analysis to quantify the mutation rates resulted from TALEN cleavage and loss of the enzyme site.

     
  5. 5.
    The output also includes a separate table containing two columns: base and score. The column “base” indicates the starting positions of target sites as in the “starting index” discussed above. The column “score” shows the highest score of TALEN pairs targeting that particular base, when there are multiple pairs starting at the same position. This table can be copied into Microsoft Excel to generate a graph (see Fig. 1). If you use brackets in the input sequence to define your base of interest as position one (seeNote5), this graph can be very informative and help you visualize high-scoring TALEN target sites near your base of interest.
    Fig. 1

    Generating a graph showing the TALEN pair composite score versus target site position. (a) Brackets are placed around a base to define it as position one of the input sequence. The table below contains the numbering of bases next to the composite scores of TALEN pairs starting at the corresponding bases. This table can be copied into a spreadsheet to generate the graph shown in (b). (b) Composite scores plotted against target site position. Adapted from [16]

     
  6. 6.

    Choose pairs of TALENs to construct from the top scoring pairs on the output list (seeNotes7 and 8) with preference to TALEN target sites closer to your target base, if applicable. As SAPTA will evolve as more information become available, please check SAPTA online tutorials for the up-to-date recommended cutoff score for active TALEN pairs. The SAPTA algorithm was developed using experimental data of TALENs constructed with the NK RVD. However, we have used SAPTA to select target sites for TALENs with NN or NH RVDs and also observed effective cleavage with these NN- or NH-TALENs.

     
  7. 7.

    It is important to limit cleavage by designed TALENs at off-target sites. To determine if there are other sites with sequence homology to the intended target site, check for potential off-target sites using PROGNOS. This link (http://baolab.bme.gatech.edu/Research/BioinformaticTools/prognos.html) also contains tutorials. Chapter 24 by Eli J. Fine et al., this volume contains more details on the off-target analysis.

     

3.2 Assembly of Homodimeric or Heterodimeric Target Plasmids

  1. 1.

    Prepare an SSA target plasmid backbone (seeNote1). In our example, we use AscI and SbfI restriction sites for directional cloning of the TALEN target sequences.

     
  2. 2.

    Digest the SSA target plasmid backbone using the two chosen restriction enzymes. Here we use AscI and SbfI to clone our targets. To ensure complete digestion, allow the digestion to proceed for at least 3 h.

     
  3. 3.

    Load the digested vector on 0.7 % agarose gel to separate away from uncut DNA and the insert that is cut out. Gel-isolate the digested plasmid backbone and use a commercially available gel extraction kit to remove the agarose.

     
  4. 4.
    Order sense and antisense oligonucleotides that contain the sequences of TALEN target sites and a spacer. The ends of the oligonucleotide pairs should be compatible with each other and with the restriction-digested ends of the SSA plasmid backbone (see Fig. 2 and Note9).
    Fig. 2

    Schematic of target plasmid assembly. Three pairs of oligonucleotides that contain the left TALEN half-site, a spacer with an EcoRI site, and the right TALEN half-site are ligated into the vector. The ends of each oligonucleotide pair are compatible with its adjacent oligonucleotide pair(s) or the backbone vector. Oligonucleotide pairs were ligated into the vector digested with AscI and SbfI resulting in a EGFP gene interrupted by a stop codon, zinc finger nuclease site and TALEN target site. Oligonucleotide pairs containing TALEN half-sites (red lines with arrows) can be used in assembling both homodimeric TALEN targets and heterodimeric TALEN targets. Various spacer lengths from 11 to 30 bp can be used in the ligation. Figure from [16]

     
  5. 5.

    Resuspend these oligonucleotides using 10 mM pH 8.0 Tris–HCl solution to a final concentration of 10 μM.

     
  6. 6.

    Kinase the single-stranded oligonucleotides in the following reaction mixture: 0.7 μl 10 μM oligonucleotide, 2.5 μl 10× T4 DNA ligase reaction buffer (NEB) (seeNote10), 2 U of T4 polynucleotide kinase (NEB), and sterile water to 25 μl. Incubate this reaction mixture at 37 °C for 40 min, and 65 °C for 20 min to heat inactivate the enzyme.

     
  7. 7.

    Mix the entire reaction mixture of the sense strand oligonucleotide with the antisense strand oligonucleotide. Heat the combined mixture to 95 °C for 10 min, and allow it to cool down at room temperature for at least 1 h. The sense and antisense oligonucleotides are now annealed and form double-stranded oligonucleotide pairs. Proceed to next step or stored the annealed pairs at −20 °C until use.

     
  8. 8.

    Ligate the left oligo pair, the spacer oligo pair, the right oligo pair (seeNote9), and the digested and gel-isolated plasmid backbone in the following reaction: 1 μl each of kinased and annealed left oligo pair, spacer oligo pair, and right oligo pair from step 7, respectively, 50 ng of digested and gel-isolated plasmid backbone from step 3, 2 μl of 10× T4 DNA ligase reaction buffer (NEB) (seeNote10), 200 U of T4 DNA ligase (NEB), and bring final volume to 20 μl with sterile water. Incubate at 16 °C overnight.

     
  9. 9.

    Transform the ligated products into chemically competent E. coli. Plate transformation onto LB plates with suitable antibiotic and incubate plates overnight at 37 °C.

     
  10. 10.
    Screen 1–3 colonies by colony PCR. Amplify using primers annealing to the plasmid backbone flanking the insert (seeNote11 and Fig. 3).
    Fig. 3

    Example gel image of colony PCR results for screening SSA target plasmid. Lane “L” is 100 bp DNA ladder (NEB). The two lanes marked by “−” are negative clones. The lane “+” contains the correct clone for an SSA target plasmid with TALEN site ligated into the vector

     
  11. 11.

    Start overnight cultures of positive clones in LB broth with suitable antibiotic. Prepare plasmids using any commercially available miniprep kit for sequence confirmation and any downstream applications (seeNote12).

     

3.3 Cell Transfection for SSA Assay

  1. 1.

    Incubate 24-well cell culture plates with 0.1 % gelatin solution that covers bottom of the wells at 37 °C for 30 min to several hours. After incubation, aspirate all the gelatin solution.

     
  2. 2.

    Seed HEK293T cells into the gelatin-treated plates at 80,000 cells per well in 500 μl 293T cell culture media.

     
  3. 3.

    3–6 h after seeding cells, prepare the following mixture per well for calcium phosphate transfection: 2.5 μl of 2 M CaCl2, 100 ng of each TALEN monomer plasmid (total 200 ng for a pair of TALENs), 10 ng of an SSA target plasmid (seeNote13), and sterile H2O to 20 μl.

     
  4. 4.

    Add 20 μl 2× HBS, pipet up and down gently for around ten times, and then completely depress plunger to blow bubbles several times.

     
  5. 5.

    Incubate the mixture for 2–5 min at room temperature. Incubation of more than 5 min may decrease the transfection efficiency.

     
  6. 6.

    Add 40 μl of the mixture drop-wise to each well of 24-well plates, and carefully return the plates to a cell culture incubator.

     
  7. 7.

    16–20 h after transfection, replace the old media with 500 μl of 293T cell culture media.

     
  8. 8.

    48 h after transfection, harvest the cells and pellet the transfected cells at 800 × g for 10 min. Gently remove the supernatant. Proceed to SSA assay or keep the cell pellets at −80 °C until use.

     

3.4 SSA Assay Using PCR

  1. 1.

    Add 70 μl of QuickExtract solution to the cell pellet from one well of a 24-well plate and pipette vigorously to completely resuspend the cells.

     
  2. 2.

    Transfer the solution to PCR tubes and run the following program in a thermal cycler to extract DNA: 68 °C for 15 min, 95 °C for 8 min, and then hold at 4 °C.

     
  3. 3.

    Use sterile water to make a fivefold dilution of the extracted DNA from step 2 (seeNote14). Votex vigorously until a homogenous solution is obtained.

     
  4. 4.

    Set up a 25 μl PCR reaction using the 2× GoTaq Green Master Mix: 5 μl of diluted DNA from step 3, 1.25 μl of each primer at 10 μM (seeNote2), 12.5 μl GoTaq Green Master Mix, and 5 μl sterile water.

     
  5. 5.

    Run the PCR as follows: 95 °C for 5 min, 35× (95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min); 72 °C for 5 min; hold at 4 °C.

     
  6. 6.

    Run 5 μl of PCR reaction on a 2 % agarose gel.

     
  7. 7.
    Quantify the percentage of SSA-repaired band in the total PCR product using the free software ImageJ or other image-analysis softwares (see Fig. 4). This percentage of SSA is a measurement of TALEN activity.
    Fig. 4

    Example SSA assay controls and results. Samples from a representative SSA assay used to quantitate the PCR products amplified from HEK293T cells. Agarose gels separated the 345-bp PCR fragments amplified from SSA-repaired target plasmids and the 514-bp PCR fragments amplified from uncut or NEHJ repaired plasmids. The percentage of the SSA-repaired products relative to the total PCR products was determined using ImageJ and shown below each lane. The negative controls cells transfected with an empty TALEN backbone and an SSA target plasmid are labeled “−”. The positive control cells transfected with a pair of GFP-ZFNs [17] and an SSA target plasmid are labeled “ZFN”. The positive control cells transfected with pEGFP plasmid and an empty TALEN backbone and are labeled “G”. Lane “L” is 100 bp DNA ladder (NEB). The “TAL” lane contains the PCR from cells transfected with a pair of TALENs and the corresponding SSA target plasmid. Adapted from [16]

     

3.5 Cell Transfection for T7E1 Mutation Detection Assay

  1. 1.

    Incubate 24-well cell culture plates with 0.1 % gelatin solution that covers bottom of the wells at 37 °C for 30 min to several hours. After incubation, aspirate all the gelatin solution.

     
  2. 2.

    Seed HEK293T cells into the gelatin-treated plates at 40,000 cells per well in 500 μl 293T cell culture media with freshly added l-glutamine at 2 mM (seeNote15).

     
  3. 3.

    One day after seeding cells, prepare 24.1 μl of the plasmid mixture which contains 550 ng of each TALEN (total 1.1 μg TALEN) and 11 ng of EGFP plasmid. Bring this mixture to room temperature before next step.

     
  4. 4.

    Add 3.4 μl Fugene HD at room temperature to the plasmid mixture from step 3. Pipette up and down for more than 15 times or vortex briefly to mix (seeNote16).

     
  5. 5.

    Incubate the mixture for 10–15 min at room temperature.

     
  6. 6.

    Add 25 μl of the mixture drop-wise to each well of 24-well plates, and return the plates to a cell culture incubator.

     
  7. 7.

    48 hours after transfection, replace the old media with 500 μl of 293T cell culture media.

     
  8. 8.

    72 hours after transfection, harvest the cells and use flow cytometer to quantify the percentage of EGFP positive cells as an indication of transfection efficiency. Pellet the cells at 800 × g for 10 min. Gently remove the supernatant. Proceed to T7E1 mutation detection assay or keep the cell pellets at −80 °C until use.

     

3.6 T7E1 Mutation Detection Assay

  1. 1.

    Add 80 μl of QuickExtract solution to the cell pellet from one well of a 24-well plate and pipette vigorously to completely resuspend the cells.

     
  2. 2.

    Transfer the solution to PCR tubes and run the following program in a thermal cycler to extract DNA: 68 °C for 15 min, 95 °C for 8 min, and then hold at 4 °C.

     
  3. 3.

    Set up a 50 μl PCR reaction using the AccuPrime Taq DNA polymerase high fidelity kit: 2 μl of DNA from step 2, 2.5 μl of each primer at 10 μM, 5 μl of 10× AccuPrime buffer II, 0.2 μl of AccuPrime Taq DNA polymerase high fidelity, 2.5 μl DMSO, and sterile water to 50 μl. For every genomic locus tested, a negative control PCR reaction using cells not treated by TALENs need to be included. This negative PCR reaction will be treated the same way as the TALEN-treated samples in the following steps.

     
  4. 4.

    Run the PCR as follows: 95 °C for 5 min, 35× (95 °C for 30 s, 60 °C for 30 s, 68 °C for 1 min); 68 °C for 5 min; hold at 4 °C.

     
  5. 5.

    To verify specific amplification, mix 5 μl of the PCR reaction with 1 μl of 6× xylene cyanol loading dye and load into a 2 % agarose gel (seeNote17).

     
  6. 6.

    Purify the PCR reaction using any commercially available PCR purification kit or other suitable methods (seeNote4).

     
  7. 7.

    Set up a reaction containing 200 ng of purified PCR product and 1.8 μl of 10× NEBuffer2 in 18 μl total volume.

     
  8. 8.

    Vortex and centrifuge briefly to mix.

     
  9. 9.

    Melt and re-anneal the DNA by placing in a thermal cycler: 95 °C for 10 min; decreasing at 0.1 °C/s down to 85 °C, hold at 85 °C for 2 min; decreasing at 0.1 °C/s down to 75 °C, hold at 75 °C for 3 min; decreasing at 0.1 °C/s down to 65 °C, hold at 65 °C for 3 min; decreasing at 0.1 °C/s down to 55 °C, hold at 55 °C for 3 min; decreasing at 0.1 °C/s down to 45 °C, hold at 45 °C for 3 min; decreasing at 0.1 °C/s down to 35 °C, hold at 35 °C for 3 min; decreasing at 0.1 °C/s down to 25 °C, hold at 25 °C for 3 min; hold at 4 °C.

     
  10. 10.

    The re-annealed DNA can be stored at −20 °C.

     
  11. 11.

    Vortex and centrifuge briefly to mix.

     
  12. 12.

    Make an enzyme master mix: every 2 μl contains 0.5 μl of T7 Endonuclease I, 0.2 μl of 10× NEBuffer2, and 1.3 μl of water.

     
  13. 13.

    Add 2 μl of the master mix to each reaction, vortex and centrifuge briefly to mix, and immediately place in a thermal cycler set for 37 °C for 60 min, with heated lid setting off.

     
  14. 14.

    After 60 min, immediately remove the reactions from the thermal cycler, centrifuge briefly, and quench by adding EDTA to a final concentration of 45 mM. This can be accomplished by adding 6.94 μl of a mixture containing a ratio of 2.45:4.49 of 0.5 M EDTA to 6× xylene cyanol loading dye.

     
  15. 15.

    Vortex and centrifuge briefly to mix.

     
  16. 16.

    Quenched reactions can be stored at 4 °C for several hours or at −20 °C indefinitely.

     
  17. 17.

    Load as much of the reactions as possible (≥25 μl) into a 2 % agarose gel that was cast with wide wells to help visualize cleavage products clearly. We use combs with tooth width of 7 mm. For each genomic site tested, load the negative control sample mentioned in step 3 next to the TALEN-treated sample for easy comparison. Run the gel until the bands are well separated.

     
  18. 18.

    When imaging the gel, make sure that the exposure time is properly adjusted so that none of the bands are saturated, as this will interfere with accurate quantification of the band intensities.

     
  19. 19.

    The free software ImageJ (NIH) can be used to quantify the bands on the gel. There are many online tutorials that detail this process. Chapter 24 by Eli J. Fine et al., this volume contains more details.

     
  20. 20.

    The percent of alleles that show evidence of nonhomologous end-joining (NHEJ) can be calculated from the band intensities according to the formulas [18] (seeNotes18 and 19):

     
$$ {f}_{cut}=\frac{ CleavageBan{d}_1+ CleavageBan{d}_2}{ CleavageBan{d}_1+ CleavageBan{d}_2+ UncleavedBand} $$
$$ \% NHEJ=100\times \left(1-\sqrt{1-{f}_{cut}}\right) $$

4 Notes

  1. 1.

    An SSA backbone plasmid should contain two repeat sequences separated by one or more stop codons, an optional control target site, and two unique restriction sites that are each present once in the plasmid. Our vector contains a ZFN target site that can be used as an activity control. The two restriction sites are used to insert TALEN target sites that include a left binding site, followed by a spacer and a right binding site.

    Our SSA backbone plasmid contains an EGFP gene, interrupted after 327 bp with a stop codon, the target site for a pair of GFP-targeted ZFNs [17], an AscI, and an SbfI cloning sites (plasmid kindly provided by Dr. Matthew Porteus, Stanford University). The downstream portion of the EGFP gene includes a 42-bp region repeating the sequence of the EGFP gene before the stop codon. The effect of varying the repeat size was compared in the chapter 22. Part of the SSA backbone sequence is shown below with the 42-bp repeats shaded in black, the ZFN target sites shaded in gray, and the cloning restriction sites underlined.

    Open image in new window

    TALEN target sites can be cloned into the backbone plasmid using the AscI and SbfI sites, indicated above. The example SSA target plasmid, below, was cloned using these sites. The bold and underlined left and right TALEN binding sites are separated by a 17-bp spacer containing an EcoRI site.

    Open image in new window

     
  2. 2.

    The forward primer for SSA PCR should be located upstream of the first 42-bp EGFP repeat (seeNote1), and the reverse primer should be located downstream of the second 42-bp repeat. The size of the PCR product should be in the range of 300–500 bp to better separate the PCR products from the original/uncut target plasmid and the SSA-repaired target plasmid on a 2 % agarose gel.

     
  3. 3.

    Primers for the T7E1 assay to amplify specific genomic loci can be designed using Primer-BLAST provided by NCBI, or similar programs. Ideally primers should have melting temperatures (Tm) around 60 °C. The PCR amplicon size should be between 300 and 600 bp because amplicons longer than 600 bp are prone to nonspecific degradation by the T7E1 enzyme. Primers should be carefully positioned so that the two cleaved bands generated by T7E1 digestion are both larger than 100 bp.

     
  4. 4.

    Any commercially available PCR purification kit can be used to clean up the PCR amplifications for the T7E1 assay. If a large number of samples need to be processed, you can alternatively use a high-throughput, magnetic-bead-based method to purify your PCR reactions (see Chapter 24 by Eli J. Fine et al., this volume for details). If nonspecific PCR products are present, gel isolation and gel extraction kit can be used to purify the band of desired size.

     
  5. 5.

    When you paste the sequence of interest into the input box on the SAPTA Web site, you may place brackets around the nucleotide you hope to target (e.g., ACGT[T]GTA). The nucleotide in brackets will be defined as position one in the output tables of the search, which allows users to more easily identify the distance of each scored TALEN target sites from the specific base of interest.

     
  6. 6.

    The frequency of high-scoring TALEN pairs in the human genome can be found in the study by Lin et al. [16]. Target sites with SAPTA scores of 35 and higher were found on average within the first 29 bp of open reading frames of 48 human genes, with a standard deviation of 32 bp.

     
  7. 7.

    Various methods can be used to construct TALENs, including those published by Zhang et al. [19], Cermak et al. [5], Briggs et al. [20], Reyon et al. [21], Schmid-Burgk et al. [22], and Kim et al. [23].

     
  8. 8.

    To ensure efficient targeting of the gene of interest, you may construct 2–3 top-ranking pairs per site. TALENs with less than 21 repeat arrays are preferred [21]. TALEN binding can be impaired by cytosine methylation in CpG islands [23, 24, 25]. When you choose target sites, you should avoid CpG dinucleotide if possible.

     
  9. 9.

    Examples of oligonucleotide pairs for making the SSA target in Note1 are shown below with each sense and antisense oligonucleotide paired. The overhangs of these oligonucleotide pairs were designed to be ligated into the AscI and SbfI digested backbone in the following order: backbone AscI site—left oligo pair—spacer oligo pair—right oligo pair—backbone SbfI site.

    Left oligo pair:

    5′-CGCGCCTCATCCACGTTCACCTTGCCCCACAGGGCAGT-3′

    3′-GGAGTAGGTGCAAGTGGAACGGGGTGTCCCGTCATAG-5′

    Spacer oligo pair (for 17 bp spacer):

    5′-ATCGATGAATTCTTAAG-3′

    3’-CTACTTAAGAA-5′

    Right oligo pair:

    5′-ACTGCCCTGTGGGGCAAGGTGAACGTGGATGACCTGCA-3′

    3′-TTCTGACGGGACACCCCGTTCCACTTGCACCTACTGG-5′.

     
  10. 10.

    T4 DNA ligase reaction buffer contains ATP which is not stable when subjected to many freeze–thaw cycles. It is recommended to aliquot this buffer into small volumes (20–100 μl depending on the volume to be used each time) and store at −20 °C.

     
  11. 11.

    To screen for positive clones of the SSA target plasmid, we amplified with a forward primer annealing about 175 bp upstream of the insert (TALEN target site) and a reverse primer annealing 130 bp downstream of the insert. Using our primers, the expected PCR product of a positive clone is ~400 bp. Since the insert is approximately 80 bp, the PCR product should be in the size range that allows discerning an 80-bp difference.

     
  12. 12.

    Sequencing the homodimeric SSA target plasmids (the left and the right TALEN binding sites are identical) is complicated by the palindromic sequence. Reads were more successful using the Power Read DNA sequencing service provided by Eurofins MWG Operon. Heterodimeric target plasmids do not have this problem and can be sequenced by any regular sequencing service.

     
  13. 13.

    To estimate transfection efficiency for the SSA assay, we transfect 200 ng of a stuffer plasmid and 10 ng of an EGFP plasmid, and use flow cytometry to quantify the percentage of GFP positive cells when cells are harvested 48 h after transfection.

     
  14. 14.

    It is difficult to pipette and dispense a small volume of the DNA extraction prepared by the QuickExtract solution due to its stickiness. We found that diluting the DNA extraction can reduce pipetting artifact and lead to more consistent results.

     
  15. 15.

    l-Glutamine can degrade over time in liquid cell culture medium. We found that adding fresh l-glutamine when cells are seeded is required for high TALEN transfection efficiency for the T7E1 assay.

     
  16. 16.

    Fugene HD reagent can only be kept in air-tight, non-silanized glass containers until mixed with plasmid solution. Prior to the mixing step, do not dilute Fugene HD or aliquot this reagent into regular microcentrifuge tubes or strip tubes made of polypropylene or other non-glass materials.

     
  17. 17.

    T7E1 assay involves DNA fragments in the 100–600 bp range. Many loading dyes contain components that run in this range, which would obscure the bands that need to be visualized. Make sure you choose a dye that does not have anything that will co-migrate with DNA fragments of that size. The xylene cyanol dye we used migrates at 800 bp to 1 kb on a 2 % agarose gel and thus will not interfere with the visualization of DNA bands in the T7E1 assay.

     
  18. 18.

    The T7E1 assay has a detection limit of ~1 % gene modification [18]. For higher sensitivity of detection, please see Chapter 24 by Eli J. Fine et al., this volume.

     
  19. 19.

    The gene-modifying efficiency of TALENs is influenced by genomic context (e.g., binding of other cellular proteins, how accessible the genomic loci are, the methylation status) at the target sites. SSA assay, on the other hand, is a method to measure the activity of TALENs without affected by genomic context. If SSA assay shows high activities of TALENs, but T7E1 assay fails to show any activity at the endogenous loci, it may indicate that some genomic context is limiting the cleavage by TALENs.

     

Notes

Acknowledgments

This work was supported by the National Institutes of Health as an NIH Nanomedicine Development Center Award (PN2EY018244 to G.B.).

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Yanni Lin
    • 1
  • Thomas J. Cradick
    • 1
  • Gang Bao
    • 1
  1. 1.Wallace H. Coulter Department of Biomedical EngineeringGeorgia Institute of TechnologyAtlantaUSA

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