Jarryd M. Campbell and Stephen C. Ekker, Mayo Clinic
Woods Hole Zebrafish Development and Genetics: TALEN Lab
Background
Transcription activator-like effector nucleases (TALENs) are proteins with a customizable DNA
binding domain and a FokI endonuclease. The TALEN DNA binding domain consists of a repeating
sequence of 33-34 amino acids with variability at 12 and 13 (a position called the repeat variable
diresidue, or RVD). Different RVDs have been found to bind each of the four nucleotides; traditionally,
NI binds adenine, HD binds cytosine, NG binds thymine, and NN or NK binds guanine. The DNA
binding domain can be assembled using this code to bind a particular sequence in the genome,
bringing with it the FokI endonuclease that will generate a double-strand break. FokI is a dimer, thus
two TALEN arms must be introduced to flank a particular region for editing.
The classic cellular response to a double strand break is collectively referred to as double-strand
break repair (DSBR) and includes non-homologous end joining (NHEJ) and homology-directed repair
(HDR). NHEJ is an error-prone process that often makes insertions or deletions (indels) at the cut
site, and thus is most commonly used to knockout a gene. HDR, which includes homologous
recombination (HR), requires and exogenous DNA “donor” that has homology to the locus from which
the cell can use as a template for repair. Unlike NHEJ, this process is not random and is usually used
for programmed knock-ins.
(Campbell et al., Circ Res, in press)
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Lab Objectives
Jarryd M. Campbell and Stephen C. Ekker, Mayo Clinic
Traditionally, gene knockouts in zebrafish by NHEJ are genotyped after injections, raised, and then
crossed to generate a stable line that can be further studied. This long-term stability of mutations in
the DNA makes TALENs fundamentally different than morpholinos. In this lab demonstration,
however, we will use a more nonconventional approach to using TALENs. The first goal of this course
is to observe a mutant phenotype after 24 hours of development by targeting the gene golden (gol).
Mutations of this gene are recessive, therefore conversion of both chromosomal copies are required
to see a phenotype. This experiment will showcase the high-activity potential of TALENs in somatic
tissue.
While performing the injections is critical to a successful TALEN experiment, equally as important is
the TALEN design. There are several rules that seem to govern TALEN activity. Several groups have
put together helpful softwares that are available online, which has simplified TALEN design for any
scientist. The second goal of this course is to use one of these softwares, Mojo Hand
(talendesign.org). Mojo Hand allows the user to design TALENs against any gene on the National
Center for Biotechnology Information (NCBI) website. We will search the NCBI website for a gene we
would theoretically like to knock-out, and then walk through how to use the software and interpret the
results.
Materials
1. mRNA encoding TALENs against golden
2. Nuclease-free water for mRNA dilution
3. Egg water or 1X E3 Medium
4. 1% agarose dishes (egg water or 1X E3 medium) for injection wells
5. 1mm glass capillary tubes to make needles (World Precision Instrument, Sarasota, FL: cat no.
1B100F-4)
6. Forceps
7. Disposable 1 uL pipets for droplet calibration
8. Glass pipet for embryo transfer
9. 10 cm petri dishes
Procedure
A) To begin, RVDs that target golden must be assembled and cloned into the TALEN scaffold. This
has already been done for you using the Golden Gate method of RVD assembly. The Golden Gate
method of assembly is described on page one of the supplemental materials. The DNA sequence
that is being targeted and the corresponding RVDs are listed here:
(Dahlem et al., PLoS Genet., 2012)
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Jarryd M. Campbell and Stephen C. Ekker, Mayo Clinic
B) Next, mRNA must be prepared from the final TALEN scaffold to inject. This has also already been
done for you. The plasmid was first linearized with the restriction endonuclease NotI, and following
linearization mRNA was transcribed using the SP6 mMessage mMachine kit (Life Technologies,
cat no. AM1340). The TAL scaffold plasmid map has been included in supplemental materials page
two for more information. Keep in mind, different TALEN scaffolds require different restriction
enzymes for linearization and different RNA polymerases for in vitro transcription.
Day 1 Procedure
C) We are going to inject TALENs targeting golden into one-cell zebrafish embryos. The procedure to
inject is identical to morpholino injection. The following steps highlight the basic steps for a TALEN
injection.
1. Each of you has an aliquot of 0.5 uL left/right TALEN mRNA (788 ng/uL) from which to dilute to
25 ng/uL using nuclease-free water. This is the concentration we will microinject.
2. After dilution, load and calibrate the needle (see “Egg Microinjection Technique and
Morpholinos” protocol)
3. Collect zebrafish embryos and transfer 20-30 to an injection plate to microinject.
4. Inject 2 nL of mRNA into the yolk or cell of the zebrafish embryos on the injection plate. This
will correspond to 50 pg of each TALEN arm, which is 100 pg of RNA per embryo.
5. Move the injected embryos to a petri dish and label the dish with your initials, the date and the
concentration of mRNA injected. Do the same for the un-injected fish.
6. Repeat steps 4-6 with injection volumes of 3 nLs and 4 nLs. When working with TALENs, it is
important to perform a dose curve such as this to understand the toxicity of the reagent. Generally,
we try and work at the LD10 (the dose at which 10% of the embryos do not survive).
Day 2 Procedure
D) The zebrafish have been allowed to develop for approximately 24 hours, so we are now going to
look for a visible phenotype characteristic of golden defects.
1. The death rate should be noted the day after injections. Count the percentage dead at each
dose. If this TALEN was going to be used again for future work, we would want to work around the
LD10. For this experiment, seeing pigment loss at any of the doses will be useful.
2. View both injected and un-injected fish under the microscope. To easily see pigment
differences, place a white piece of of paper under the dish while on the microscope. Below are
examples of pigment changes, primarily of the eye. Identify pigment changes. Here are a range
of examples at two different doses (25 pg, left; 100 pg, right).
The red arrows indicate a wild type phenotype, in which there is dark pigmentation in the eye and
along the body. The black arrows point to areas of pigment loss that is characteristic of gol mutants.
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Jarryd M. Campbell and Stephen C. Ekker, Mayo Clinic
Remember that any changes we see by TALENs is at the level of DNA, unlike morpholinos that
impact RNA. In wild type fish, pigmentation comes on slowly but is noticeably dark in color. However,
gol knockdown or knockout fish will have a much more delayed pigment onset and appear clear or
white. Any completely clear/white fish that are seen are bi-allelic knockouts, meaning both alleles are
mutated in most cells of the entire organism. A much more common phenotype will be fish that have
patches of light and dark pigment. This is a characteristic of mosaicism, in which only a subset of
cells have mutated alleles.
E) For any scientist interested in TALEN technology, tips for choosing a sequence to target is going to
be helpful. In principle, TALENs should be able to target any sequence; however, best-practice
guidelines have been published to narrow down the number of potential sites and to improve the
chances of higher activity. In general, the rules that govern TALEN activity appear to be similar
across scaffolds.
These rules have been incorporated into several easy-to-use softwares available online. For this
class, we will focus on Mojo Hand (talendesign.org).
1) Log onto the NCBI website and write down the gene ID of your favorite gene
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Log onto http://www.talendesign.org
2) Enter your email and the gene ID
Jarryd M. Campbell and Stephen C. Ekker, Mayo Clinic
4) Press “Run TALEN Script”
5) While the program is running, you will see this. This may take a few minutes, depending on the
size of the gene.
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Jarryd M. Campbell and Stephen C. Ekker, Mayo Clinic
6) Eventually you will get your results. Select “TALEN Sites & Enzymes”
7) Finally, you will have the Results page.
The results page gives you a lot of information. At the top of each panel, you will see a name with
“_E#”. This is telling you the gene name the program pulled from NCBI, as well as the exon being
targeted. The script knows exon/intron differences by accessing this information from the NCBI
website. TAL1 and TAL2 correspond to the sites being targeted (TAL2 is targeting the reverse
complement, also shown), and their corresponding RVD sequences are listed below. At the bottom of
each panel will be a list of enzymes. The purpose of this software is to identify restriction enzyme
sites in the spacer region (sequence between the TALEN pairs) that can be used to easily genotype
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Jarryd M. Campbell and Stephen C. Ekker, Mayo Clinic
using a PCR followed by restriction digest approach. The enzyme list is taken from any number of
companies, but the default is NEB.
If you choose “Download as CSV file,” you will get the same information in Excel or a similar program
for ease of organization. “Save as candidate” will send an email to the account you entered with the
same information.
Supplemental Materials
The Golden Gate Method of RVD assembly from Cermak, et al., Nucleic Acids Res., 2011
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Supplemental Materials
Jarryd M. Campbell and Stephen C. Ekker, Mayo Clinic
Here are the plasmid maps of the TAL scaffolds from Dahlem, et al., PLoS Genet., 2012 we will use
for this course. Keep in mind, the RVDs are cloned between the Esp3I sites (Esp) by the Golden Gate
method of assembly. The NotI site (Nt) is used to linearize the plasmid, and an SP6 RNA polymerase
is used to make artificial sense RNA for injections.
Procedure for making RNA:
1) Linearize TALEN scaffold with NotI
2) Use SP6 mMessage mMachine kit for in vitro runoff transcription
3) Purify, quantify and inject!
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