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

Summary

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.

Abstract

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.

Introduction

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 example^1-3). 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....

Protocol

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 genomic sequence into the box labeled "Sequence".
   2. Within the "Provide Custom Spacer/RVD Lengths" tab select the spacer length....

Results

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.......

Discussion

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 studies^1,21,23 and can.......

Materials

List of materials used in this article

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 (1,000x 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