Name: genome editing in astyanax mexicanus using transcription activator-like effector nucleases (talens)
Uploaded: 2016-06-20T14:00:00
Description: Watch this Scientific Journal Video about Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs at JoVE.com

This work was funded by the Department of Genetics, Development and Cell Biology and Iowa State University and by NIH grant EY024941 (WJ).Dr. Jeffrey Essner provided comments on the manuscript.

All animal procedures were in accordance with the guidelines of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee at Iowa State University and the University of Maryland.
1. TALEN Design
<ol>
	<li>Input desired target sequence to a TALEN design website. (For example: <a href="https://tale-nt.cac.cornell.edu/node/add/talen">https://tale-nt.cac.cornell.edu/node/add/talen</a>). Input chosen spacer/repeat array lengths.
	<ol>
		<li>Copy the ge...

Great strides have been made in recent years towards understanding the genetic basis of the evolution of traits. While candidate genes underlying the evolution of a number of traits have been identified, it has remained challenging to test these genes in vivo due to the lack of genetic tractability of most evolutionarily interesting species. Here we report a method for genome editing in A. mexicanus, a species used to study the evolution of cave animals. Genetic mapping studies1,21,23 and can...

Understanding the genetic basis of trait evolution is a critical research goal of evolutionary biologists. Considerable progress has been made in identifying loci underlying the evolution of traits and pinpointing candidate genes within these loci (for example1-3). However, functionally testing the role of these genes has remained challenging as many organisms used for studying the evolution of traits are not currently genetically tractable. The advent of genome editing technologies has greatly increased genetic manipulability of a wide range of organisms. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) have been used to generate targeted mutations in genes in a number of organisms (for example4-11). These tools, applied to an evolutionarily relevant system, have the potential to revolutionize the way evolutionary biologists study the genetic basis of evolution.
Astyanax mexicanus is a species of fish that exists in two forms: a river-dwelling surface form (surface fish) and multiple cave-dwelling forms (cavefish). A. mexicanus cavefish evolved from surface fish ancestors (reviewed in12). Populations of cavefish have evolved a number of traits including loss of eyes, decrease or loss of pigmentation, increased numbers of taste buds and cranial neuromasts, and changes in behavior such as loss of schooling behavior, increased aggression, changes in feeding posture and hyperphagia13-19. Cavefish and surface fish are interfertile, and genetic mapping experiments have been performed to identify loci and candidate genes for cave traits1,20-26. Some candidate genes have been tested for a functional role in contributing to cave traits in cell culture1,19, in model organisms of other species21 or by overexpression27 or transient knockdown using morpholinos28 in A. mexicanus. However, each of these methods has limitations. The ability to generate mutant alleles of these genes in A. mexicanus is critical for understanding their function in the evolution of cavefish. Thus, A. mexicanus is an ideal candidate organism for application of genome editing technologies.
Here we outline a method for genome editing in A. mexicanus using TALENs. This method can be used to evaluate mosaic injected founder fish for phenotypes and for isolating lines of fish with stable mutations in genes of interest29.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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			<td height="21" style="height:21px;width:216px;">Equipment</td>
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			<td height="21" style="height:21px;width:216px;">Thermocycler</td>
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			<td height="21" style="height:21px;width:216px;">Needle puller</td>
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			<td height="84" style="height:84px;width:216px;">Note: As far as we know, supplies from different companies can be used unless otherwise indicated</td>
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			<td height="42" style="height:42px;width:216px;">Golden Gate TALEN and TAL Effector Kit 2.0</td>
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			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
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			<td height="126" style="height:126px;width:216px;">BsaI</td>
			<td style="width:71px;">New England Biolabs (ordered through Fisher)</td>
			<td style="width:119px;">50-812-203</td>
			<td>Use BsaI, not BsaI-HF (as described in the Golden Gate TALEN and TAL Effector Kit protocol)</td>
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			<td style="width:119px;">provided with restriction enzymes</td>
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			<td height="84" style="height:84px;width:216px;">10x T4 ligase buffer</td>
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			<td>Other Taq can be used, but the reaction should be adjusted accordingly</td>
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			<td height="126" style="height:126px;width:216px;">Quick ligation kit</td>
			<td style="width:71px;">New England Biolabs (ordered through Fisher)</td>
			<td style="width:119px;">50-811-728</td>
			<td>We use Quick Ligase for all TALEN assembly reactions</td>
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			<td height="42" style="height:42px;width:216px;">One Shot TOP10 Chemically Competent E.coli</td>
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			<td>Other chemically competent cells or homemade competent cells can be used</td>
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		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Esp 3I</td>
			<td style="width:71px;">Thermo Sci Fermentas Inc (Ordered through Fisher)</td>
			<td style="width:119px;">FERER0451</td>
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			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">27106</td>
			<td>The Qiagen kit should be used for the initial plasmid preparation (as described in the Golden Gate TALEN and TAL Effector Kit protocol)</td>
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			<td height="21" style="height:21px;width:216px;">QIAquick PCR Purification Kit</td>
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			<td style="width:119px;">E-3119-125</td>
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			<td height="84" style="height:84px;width:216px;">Sac I</td>
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			<td style="width:119px;">PR-R6061</td>
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			<td style="width:119px;">AM1348M</td>
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			<td height="21" style="height:21px;width:216px;">Rneasy MinElute Cleanup Kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">74204</td>
			<td></td>
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			<td height="84" style="height:84px;">NorthernMax-Gly Sample Loading Dye&#160;</td>
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			<td style="width:119px;">AM8551</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Eliminase</td>
			<td style="width:71px;">Decon (ordered through Fisher)</td>
			<td style="width:119px;">04-355-32</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fisherbrand Disposable Soda-Lime Glass Pasteur Pipets</td>
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			<td height="84" style="height:84px;width:216px;">Standard Glass Capillaries</td>
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			<td style="width:119px;">1B100-4</td>
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			<td height="84" style="height:84px;width:216px;">Microcaps</td>
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			<td style="width:119px;">1-000-0010</td>
			<td></td>
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			<td height="84" style="height:84px;width:216px;">Eppendorf Femtotips Microloader Tips for Femtojet Microinjector</td>
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			<td style="width:119px;">E5242956003</td>
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			<td height="21" style="height:21px;width:216px;">Sodium hydroxide</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">S318-500</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris base</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP152-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome editing in astyanax mexicanus using transcription activator-like effector nucleases (talens)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

TALEN pair injections result in binding of the RVDs to specific DNA nucleotides and thus dimerization of FokI domains, resulting in double stranded breaks39 which can be repaired through non-homologous end joining (NHEJ). NHEJ often introduces errors that result in insertions or deletions (indels). Indels can be identified by amplifying the region surrounding the TALEN target site and digesting the resulting amplicon with a restriction enzyme that cuts within the TALEN...

Watch this Scientific Journal Video about Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs at JoVE.com

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

The overall goal of this procedure is to introduce mutations into specific genes in the cave fish Astyanax mexicanus. This method can help answer key questions in the evolutionary biology field about the genetic basis of the evolution of cave traits. The main advantage of this technique is that the effect of loss of the specific gene can now be tested in Astyanax mexicanus.After designing and assembling transcription activator-like effector nucleases, or TALENs, according to the text protocol, use two microliters of Sac1 to digest four micrograms of sequence verified template at 37 degrees Celsius for two hours. Run two microliters of the Sac1 digested plasmid on a one point five percent agarose gel and check for a single band to verify digestion. To carry out T three mRNA production, use zero point five micrograms of linearized template to set up half reactions following a standard protocol.Incubate at 37 degrees Celsius for two hours. Then add zero point for microliters of DNase and incubate at 37 degrees Celsius for 15 minutes. After purifying the mRNA, to check the quality, use a product that eliminates RNase contamination to clean the gel apparatus.Then, prepare a one point two percent gel. Mix one microliter of the mRNA, four microliters of nuclease-free water, and five microliters of glyoxal loading dye. Incubate the samples at 50 degrees Celsius for 30 minutes.Then briefly centrifuge the tubes, then place them on ice before running the gel. After checking the concentration the mRNA, prepare aliquots before storing at minus 80 degrees Celsius. Refer to the text protocol for additional details.After preparing injection needles and plates according to the text protocol, induce spawning in A.mexicanus by overfeeding fish for three to four days prior to breeding and place the fish into fresh water. Raise the water temperature two degrees Fahrenheit to approximately 76 degrees Celsius. Every 15 minutes, check for surface fish eggs in the dark and transfer them into glass bowls to prevent them from sticking to the surface.Hundreds of eggs can be obtained from a single pair of surface fish. Under a microscope, sort the isolated eggs and transfer those at the one cell stage to fresh system water. Load the diluted mRNA into the back of the needle and detach the needle to a microinjector.Then, using forceps, break the needle. To calibrate the needle, inject 10 times and collect the resulting drop in a microcapillary. The drop should fill a 10 by 100 disposable one microliter, 32 millimeter microcapillary to zero point five millimeters per one point five nanoliter injection.Now, insert the needle into the single cell and inject the mRNA. Collect the injected embryos in glass bowls and incubate them at 23 to 25 degrees Celsius. Remove dead embryos regularly for the first few days following injection.Record the number of dead and deformed embryos from uninjected control and injected plates. Refer to the text protocol for additional details. After euthanizing fish according to your institutions IACUC protocol, to extract the DNA, collect the embryos into 100 microliters of 50 millimolar of sodium hydroxide and incubate at 95 degree Celsius for 30 minutes.Then cool to four degrees Celsius. Add a one tenth volume of one molar tris HCL ph 8 to the cooled embryos. Then carry out PCR using primers, for example for the oculocutaneous albinism two or OCA2 gene.For individual embryos, one microliter of DNA is sufficient for the PCR reaction. To genotype the OCA2 locus, use the restriction enzyme DSR1 to digest the resulting PCR product by adding zero point five microliters of enzyme directly to twelve point five microliters of the completed PCR reaction. After incubating at 65 degrees Celsius for two hours, run the reaction and a sample of undigested PCR product on a one point five percent agarose gel.Carry out additional analyses according to the text protocol. TALEN injections can result in double stranded DNA breaks that can be repaired by non-homologous end joining but which can also introduce insertions or deletions, or INDELs. Using PCR end restriction digestion to target the TALEN spacer region INDELs can be identified by the presence of a restriction enzyme resistant band.TALEN injections can produce biallelic gene mutations in A.mexicanus and thus some phenotypes can be assessed in founder fish. For example, OCA2, the gene hypothesized to be responsible for albinism in cave fish, was evaluated. As shown here, albino patches in OCA2 TALEN injected fish were identified that were not present in uninjected fish.In this gel image, 306 base pair PCR products from exon nine of OCA2 of A.mexicanus, were examined for loss of the restriction enzyme site in pools of 10 F1 fish from an injected founder fish. The amplicon from a control embryo was digested while a portion of the amplicon was resistant to restriction digest in F1 embryos from injected fish. In addition, the restriction digest resistant bands have been shown to contain INDELs.Once mastered, these injections can be done in less than a few hours if they're performed properly. While attempting this procedure, it's important to remember to work carefully with RNA to prevent degradation and to inject during the one cell stage. Following this procedure, phenotypic analysis can be performed in order to answer additional questions such as if a particular gene underlies a key fish phenotype when mutated in surface fish.This technique will pave the way for researchers in the field of evolutionary biology to characterize the role of candidate genes in the evolution of the cave fish Astyanax mexicanus. After watching this video, you should have a good understanding of how to inject Astyanax mexicanus embryos with TALEN mRNA.

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