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Biology

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

Published: June 20th, 2016

DOI:

10.3791/54113

1Genetics, Development and Cell Biology, Iowa State University, 2Department of Biological Sciences, University of Cincinnati, 3Department of Biology, University of Maryland

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.

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.

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 pal....

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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

  1. Input desired target sequence to a TALEN design website. (For example: https://tale-nt.cac.cornell.edu/node/add/talen). Input chosen spacer/repeat array lengths.
    1. Copy the ge.......

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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.......

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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.......

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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.

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Name Company Catalog Number Comments
Equipment
Thermocycler
Injection station
Gel apparatus
Needle puller
Nanodrop
Name Company Catalog Number Comments
Supplies
Note: As far as we know, supplies from different companies can be used unless otherwise indicated
Golden Gate TALEN and TAL Effector Kit 2.0 Addgene Kit #1000000024
Fisher BioReagents LB Agar, Miller (Granulated) Fisher BP9724-500
Fisher BioReagents Microbiology Media: LB Broth, Miller Fisher BP1426-500
Teknova TET-15 in 50% EtOH Teknova (ordered through Fisher) 50-843-314
Spectinomycin Dihydrochloride, Fisher BioReagents Fisher BP2957-1
Super Ampicillin (1000x solution) DNA Technologies 6060-1
ThermoScientific X-Gal Solution, ready-to-use Thermo Sci Fermentas Inc (Ordered through Fisher) FERR0941
IPTG, Fisher BioReagents Fisher BP1620-1
Petri dishes Fisher 08-757-13
BsaI New England Biolabs (ordered through Fisher) 50-812-203 Use BsaI, not BsaI-HF (as described in the Golden Gate TALEN and TAL Effector Kit protocol)
BSA New England Biolabs provided with restriction enzymes
10x T4 ligase buffer Promega (ordered through Fisher) PR-C1263
GoTaq Green Master mix Promega (ordered through Fisher) PRM7123 Other Taq can be used, but the reaction should be adjusted accordingly
Quick ligation kit New England Biolabs (ordered through Fisher) 50-811-728 We use Quick Ligase for all TALEN assembly reactions
One Shot TOP10 Chemically Competent E.coli Invitrogen C4040-06 Other chemically competent cells or homemade competent cells can be used
Esp 3I Thermo Sci Fermentas Inc (Ordered through Fisher) FERER0451
Plasmid-Safe ATP-dependent DNase Epicentre (Ordered through Fisher) NC9046399
QIAprep Spin Miniprep Kit Qiagen 27106 The Qiagen kit should be used for the initial plasmid preparation (as described in the Golden Gate TALEN and TAL Effector Kit protocol)
QIAquick PCR Purification Kit Qiagen 28104
GeneMate LE Quick Dissolve Agaraose BioExpress E-3119-125
Sac I Promega (Ordered through Fisher) PR-R6061
mMESSAGE mMACHINE T3 Transcription kit Ambion AM1348M
Rneasy MinElute Cleanup Kit Qiagen 74204
NorthernMax-Gly Sample Loading Dye  Ambion (ordered through Fisher) AM8551
Eliminase Decon (ordered through Fisher) 04-355-32
Fisherbrand Disposable Soda-Lime Glass Pasteur Pipets Fisher 13-678-6B
Standard Glass Capillaries World Precision Instruments 1B100-4
Microcaps Drummond Scientific Company 1-000-0010
Eppendorf Femtotips Microloader Tips for Femtojet Microinjector Eppendorf (ordered through Fisher) E5242956003
Sodium hydroxide Fisher S318-500
Tris base Fisher BP152-1

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