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="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 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 style="width:119px;">PR-C1263</td>
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			<td>Other Taq can be used, but the reaction should be adjusted accordingly</td>
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			<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>
			<td style="width:71px;">Invitrogen</td>
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			<td>Other chemically competent cells or homemade competent cells can be used</td>
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			<td height="147" style="height:147px;width:216px;">Esp 3I</td>
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			<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 style="width:119px;">E5242956003</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 ...

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