TALEN expression vectors
Yasunori Sasakura 7th May 2018
Modified on 26th January 2023
(Caution: These days our group uses TALENs much more frequently than
CRISPR/Cas9. We provide the protocols only for TALENs now.)(Figure 1 below shows the overview of this protocol.)1) TALEN kit
Currently the recommended TALEN construction kit is the Platinum Gate TALEN
kit (Sakuma et al., 2013, Scientific Reports 3, 3379). You can purchase the basic setof this kit from Addgene.
https://www.addgene.org/kits/yamamotoplatinumgate/?gclid=Cj0KCQjw4eXPBRCtARIsADvOjY2SQXgDuHaRgHLpuLY
9KyyKmIrT2J5oaUniSiFJ33H75cE7qE2re6EaAmfPEALw_wcB
2) Design of TALENs
After you select a gene to knock out, determine the region of the gene in which to
introduce mutations. We usually choose the region corresponding to the core domain
critical to the protein function, such as a DNA-binding domain for transcription
factors or a kinase domain for kinases. A transmembrane domain can also be a goodtarget for transmembrane proteins. If such a domain is not present or is obscure inyour protein encoded by the target gene, then we recommend targeting around thestart codon of the gene. Note that there might be several translational start sites or
cryptic start sites in a gene. In such cases, the efficiency of yielding phenotypes
would be reduced when you target the site around the start codon.
Once you choose the DNA region for targeting, copy and paste the DNA region (200nt long or a single exon is usually sufficient; a long DNA region will yield too many
TALEN candidates) onto TALEN targeter 2.0 (https://talent.cac.cornell.edu/node/add/talen). We use these parameters:
Select the “Provide Custom Spacer/RVD Lengths” tab.
Minimum spacer length: 15
Maximum spacer length: 18
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Minimum repeat array length: 16
Maximum repeat array length: 17
G substitute: NN
Filter options: Show all TALEN pairs (including redundant TALENs)
Streubel et al. guidelines: On
Pre-loaded sequence: default (-none-)
Scoring matrix: Default (Doyle et al.)Upstream base: Default (T only)
After entering the parameters, push the Submit button.
The program will identify several pairs of TALENs that may be good for knocking
out the gene. Paste the results in an Excel spreadsheet, then select one or two pairs
for construction. For a historical reason, we prefer to choose the hetero-lengthened
pairs in which one side has 16 repeats and the other side 16 or 17.
A TALEN pair selected in this manner has ~70% probability of being a good
TALEN pair. Therefore, we usually create two pairs for a single target to assure thesuccess of knockouts.
3) First assembly
The selected TALENs will appear as follows:
NI NG NN NN NN NG NN HD NN NI NN NI NI HD NI HD NG (Left repeat)
NN NN NN NG NG HD NI NG NN NN NI HD NG NG NG NG (Right repeat)
T ATGGGTGCGAGAACACT acaggattgctcgtatgt AAAAGTCCATGAACCC A(The DNA sequence of the target site of the above TALENs. Lowercase letters arespacers. The first and last nucleotides are zero repeat/upstream bases that are notincluded in the TALEN array.)2

Separate the array data as follows:
NN NN NN NG =a3a cassette
NG HD NI NG =a3b cassette
NN NN NI HD =a3c cassette
NG NG NG=b3 or b4 cassette (this example is a b3)
NG=last repeat
In the first assembly, it is necessary to create the a3a, a3b, a3c, and b3/b4 plasmids
having three or four aligned repeats as described above. The method for the first
assembly is completely the same as the one described in the kit, so we will not
mention it here. The first assembled clones can be shared between TALENs.
4) Second assembly
Once you obtain the a3a, a3b, a3c, and b3/b4 plasmids that are compatible with yourTALENs, then ligate the first assembled repeats to make full TALENs.
After the assembly, you have to check the activity of your TALENs. For this reason,
the second assembly should use the pBSEF1a>SanTAL::2A::mCherry construct as
the backbone vector, because the assembled TALENs can be expressed under thecontrol of the EF1a promoter, which is a good ubiquitous promoter for testing theactivity of TALENs in Ciona.
The protocol of the second assembly is as follows:
Adjust the concentration of the first assembled clones to 50-100 ng/ul.
Make a 1:1:1:1 mixture of the first clones.
Make the following mixture:
First clone mixture
50 ng/ul backbone vector
2x QuickLigase buffer
Esp3I (Fermentas)
QuickLigase (NEB)

1.0 ul
0.3 ul
3.6 ul
0.2 ul
0.2 ul3

Water

1.9 ul
7.2 ul10-13 cycles of 5 min at 37oC, 10 min at 16 oC (use a thermal cycler).
Increasing the cycle number will yield more colonies.
After the cycling is over, then add the following materials in the reaction mixture:
10x Tango buffer(Supplied with Esp3I)
20 mM DTT
Water
Esp3I

1.0 ul
1.0 ul
0.6 ul
0.2 ul30-60 min at 37 oC
5 min at 80 oC to inactivate Esp3I
This reaction will reduce the number of background colonies.
Transform E. coli and plate on an LB-amp plate.
We use handmade E. coli (titer is 107-108 colony forming units/ug DNA). Usually, 1
ul of reaction mixture yields a sufficient number of colonies.
5) Insert check
The insert check of the second assembled clones should be done with the following
primers and PCR conditions.
TALE-F: GCACCCCTCAACCTGACCCCAG
TALE-R: CTCGAAAGCTGGGCCACGATTG
PCR conditions:
5 min at 95 oC
30 cycles of 94-30s, 65-30s, 72-50s
5-10 min at 72 oC
4-10 oC4

Successfully assembled colonies will show a smear band (see the photo below). Pickup the clones and culture them in 2 ml LB-amp liquid.

Example of an insert check (from the left): first lane, marker; 2-6 lanes, TALEN
clones. Lanes 2, 3, 5, and 6 suggest the success of the assembly. The fourth lane willbe the wrong clone; do not pick the colony.

Miniprep using your laboratory method.
Check the plasmids with restriction enzyme NruI. The NN (G) repeats have one
NruI site, and you can confirm the success of the assembly based on the positions of
the NN modules. The photo below is an example of three different TALENs
digested by NruI. The second assembly does not usually fail.
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First lane: 700+300
Second lane: Promoter band+600+200
Third lane: 600+200 (in base pairs)
Fourth lane: Marker (pBluescript digested with HaeIII)
For each lane, the top band is the backbone vector. Please ignore it.
6) Checking activity
Miniprepped EF1a>TALEN::2A::mCherry plasmids are sufficient for checking its
mutation activity. In our case, the method is as follows:
Elute miniprepped DNA in 50 ul elution solution.
4 ul of plasmid solution will be used for the NruI check.
Use the remaining 10 ul of plasmid solution for electroporation.
Mix L and R plasmids. The mixture will be used for the single round ofelectroporation. For details, see Yoshida and Treen (2018).
6

Collect embryos that exhibit bright mCherry fluorescence. The shape of the embryos
may be bad, but it does not influence on the estimation of mutation rate.
Spin down the embryos and discard the seawater.
Isolate genomic DNA with the Wizard genomic DNA purification kit (Promega).
Add 600 ul nuclei lysis solution. Incubate for 1-2 hours at 50 oC, preferably withrotation.
Cool to room temperature.
Add 200 ul protein precipitation solution. Incubate on ice for 5 min.
Centrifuge at 13,500 rpm for 4 min at 4 oC.
Collect the supernatant into a new tube.
Centrifuge for 13,500 rpm for 4 min at 4 oC.
Collect supernatant into a new tube.
Add 1 ul glycogen (Roche).
Add 600 ul isopropanol.
Centrifuge for 1 min at room temperature.
Discard supernatant by decantation. Pellets may be invisible.
Add 100 ul 70% ethanol.
Centrifuge 1 min at room temperature.
Discard 70% Ethanol and dry.
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Add 10-20 ul water.
PCR amplification of the TALEN target site.
Genome DNA
10x PCR bufferdNTPs
10 pmol/ul primer
10 pmol/ul primer
ExTAQ hot-start version
D3W

1.0 ul
2.0 ul
1.6 ul
2.0 ul
2.0 ul
0.2 ul11.2 ul
20.0 ul

5 min at 95 oC, 50 cycles of (94 oC-30 s, 55 oC-30 s, 72 oC-1~3 min) 5-10 min at 72
oC, 10oC thereafter.

Electrophorese 5 ul of PCR mixture on 15% polyacrylamide gel (AA:Bis = 29:1).
The voltage should be a constant 100 V. Higher voltage will reduce the resolution of
the bands.
If mutations are introduced, shifted bands will appear. The typical examples can beseen in our knockout database(http://marinebio.nbrp.jp/ciona/sequenceFile/knockOut/gsTalens/Raldh2/Raldh2HMA.pdf).
When the mutation bands appear, subclone the bands into a conventional vector forsequencing analysis. A 70% or higher mutation frequency will be OK for further
analyses.
7) Switching vectors
When you obtain a TALEN pair that has a good mutation frequency, you may haveto transfer the repeat arrays to appropriate vectors for further analyses. There aremainly two ways to transfer them: expression vectors for tissue-specific expression
and mRNA synthesis vectors.
8

The easiest way is to repeat the second assemblies on the appropriate vectors.
Because the Golden Gate method is reproducible and highly efficient, the assemblyis usually successful. However, there is a possibility that new TALENs areincorrectly assembled in which case that the incorrectness cannot be checked
completely due to the repeat structure. For this reason, we recommend the following
methods.
7-1) Repeat switching method
Isolate assemblies from EF1a>TALEN vectors with SalI and BamHI.
Miniprepped solution
10x T buffer (Takara)
SalI
BamHI
Water

5 ul
3 ul
1 ul
1 ul
to 20 ulInverse PCR acceptor vectors with the following primers:
ACCAACAGAAGGATCCCCGAGAGGACATCACATCTGCGCGACTGTCGACCTCACTTTGGGCTTGATC(Use a high-fidelity DNA polymerase. We use PrimeStar HS DNA polymerase from
TAKARA Bio. 30 cycles is sufficient).
Add 1 ul of DpnI restriction enzyme (Takara) to the PCR solution after PCR isfinished. This enzyme digests the template vector that are methylated in E. coli.
Extract the gel from the restriction-digested plasmids and PCR products. The
restriction fragments of TALEN repeats (approx. 2,000 bp) do not separate wellfrom other DNA bands (see the photos below). Don’t worry about this, because the
In-fusion reaction is highly efficient and highly specific.

9After elution, mix the DNAs for the In-fusion reaction.
The In-fusion reaction almost always successfully yields appropriate vectors.
However, the success rate on the pHTB-TALEN vector is lower than that of othervectors, and a few nucleotides are sometimes lost at the joining site. We check thesuccess of the In-fusion reaction by sequencing with the following primers:
AATGGACTATAAGGACCAC (5’ junction site)
TTTTCGTGATCCACCGAGATGC (3’ junction site)7-2) Promoter switching method
The promoter region of the TALEN expression vector can be removed with NotI. A
new promoter can be inserted at the NotI site to switch the promoter for tissuespecific expression.
Our TALEN expression vectors were constructed on pBluescript, which is different
from that used in our pSP vector series. Therefore, promoter fragments for pSP
vectors are not compatible with pBluescript. If you wish to isolate a promoter for
TALEN expression from the pSP vector, amplify the promoter with the following
primer set:
CCGAGGGTGGCGGCCTAGGTGACACTATAGAACTC

10pSP-TALEN-promoter-switch-XhoF

CCGAGGGTGGCGGCCCTGCAGGTCGACTCTAGAggatc

pSP-TALEN-promoter-switch-BamF

TAGTCCATTGCGGCCGCAAGGGGATCpSP-TALEN-promoter-switch-R

The above shows two forward primers. When the promoter is inserted into the
BamHI/BamHI site of the pSP vector, use pSP-TALEN-promoter-switch-BamHF.
When the promoter is inserted in the XXXX/BamHI site (where XXXX is anarbitrary restriction site that is located 5’ upstream from the BamHI site), use pSPTALEN-promoter-switch-XhoF.
The PCR fragments have the homology arm for the In-fusion reaction for the NotI
site of pBS-TALENs.
(Caution: The forward primer cannot be used for a few TALEN vectors. In this case,the homology arm should be 5’-ACCGCGGTGGCGGCC-3’.)11

First Assembly of TALEN repeats Day 1
NI

NN

HD

・Mix repeat units.
・Golden Gate reaction.
・Post digestion of unassembled plasmids.
・Transformation overnight.

NG

Day 2
・Insert check.
・Incubate appropriate clones.
NNHD NI NG
a3a vector

NN NGNG HD
a3b vector

HD HD NN NI
a3c vector

NG NI HD HD
b3/b4 vector

Day 3・Mini-prep.
・(Optional) Sequencing to confirm theassembly.
etc...

There are approx. 1,000 combinations of the first assembled clones for creating
TALENs with 16~17 repeats.
→We can skip this step when we finish construction of the 1,000 clones.
＊Our group recommends using the Platinum TALEN kit (Sakuma et al., 2013).

Second Assembly of TALEN repeats
NNHD NI NG
a3a vector

NN NGNG HD
a3b vector

HD HD NN NI
a3c vector

NG NI HD HD
b3/b4 vector+

N ter lacZ C ter
TALEN cDNA

Day 1
・Mix the first assembled clones.
・Golden Gate reaction.
・Post digestion of unassembled plasmids.
・Transformation.
Day 2
・Insert check.
・Incubate appropriate clones.
Day 3
・Mini-prep.
・Perform restriction digestion to confirm theassembly.
・(optional) sequencing of 5’ and 3’ ends ofassembly.

N ter NNHD NI NG NN NGNG HD HD HD NN NI NG NI HD HD C ter
Full TALEN cDNA (approx. 3,000bp)*Once the repeat is assembled, you can replace it in any TALEN backbonevector withrestriction enzymes.

Checking mutation efficiency
of designed TALEN
EF1a promoter N ter lacZ C ter 2A mCherry
Ubiquitous promoter>TALEN::2A::mCherry

After assembly, electroporate into embryos.
We can see theexpression of TALENs
by red fluorescence.
CelI assay

・2nd assembly of your designed
TALEN into the backbone
vector of TALEN with the EF1α
promoter.
・ Miniprep and electroporation of
the vectors.
・ Expression of TALENs can bemonitored with the fluorescence ofmCherry.
・ Collect tailbud embryos withubiquitous mCherry fluorescence.

PAGE・ Genome isolation and PCRamplification of the target site.
・ Digestion with CelI or
polyacrylamide gel electrophoresis.

・(Optional) cloning of PCR bands
and sequencing to see the efficiency
Checking the occurrence of mutations by electrophoresis of mutations.

Tissue-specific TALEN vector series
promoter

N ter lacZ C ter 2A mCherry

Tissue-specific promoter>TALEN::2A::mCherry

Mesenchyme

・Insert your repeats into a
TALEN backbone vector
with a tissue-specific promoter.

Endoderm

・Expression of TALENs can bemonitored with the fluorescenceof mCherry.

After assembly, electroporation into embryos.
Epidermis

Muscle・Tissue-specific disruption ofgenes gives us important
information for elucidating genefunctions. TALENs can be used
for this purpose easily, because
they are gene-encoded proteins.

・We have already createdvectors for epidermis, muscle,neural tissue, endoderm, andmesenchyme. These vectors areavailable from the NBRP project

TALEN mRNA for knockout with reduced mosaicism and germline mutations
UTRs of tubulin gene of H. roretzi.
N ter lacZ C ter 2A mCherry
pHTB TALEN::2A::mCherrymRNA synthesis and microinjection

・Microinjected mRNA disperses
rapidly in eggs. Therefore, TALENmRNA is likely to be uptaken bymost cells of an embryo.
・Insert your repeats into the
backbone vector of TALEN with
UTRs to increase translation
efficiency.
・In vitro transcription of mRNA isnecessary after construction.

%

Mutation frequency
in sperm genome

animal・This method enables you to
introduce mutations in a germ cell
genome as well as a somatic cell
genome.