基因组编辑在<em> Astyanax mexicanus</em>使用转录因子样效应核酸酶（TALENS）
Summary
基因靶向诱变现在有可能在宽的范围内使用的基因组编辑技术生物体。在这里，我们演示了有针对性的基因突变的协议使用转录激活像Astyanax mexicanus，鱼品种，包括面鱼和cavefish效应核酸酶（TALENS）。
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
了解性状进化的遗传基础是进化生物学家的一个重要研究目标。相当大的进展在查明位点特征的演变和背后这些位点（例如1-3）内精确定位候选基因进行了。然而，在功能上测试这些基因的作用仍然具有挑战性用于研究性状的进化因为许多有机体当前不是遗传容易处理。基因组编辑技术问世已经大大增加了广泛的生物体的遗传操作性。转录活化剂-样效应核酸（TALENS）和群集定期相互间隔短回文重复序列（CRISPRs）已被用来产生在一个数生物体（例如4-11）在基因靶向的突变。这些工具，应用到进化相关制度，有潜力彻底改变进化生物学家研究进化的遗传基础。
Astyanax mexicanus是鱼类的物种存在两种形式：多穴居形式（cavefish）河居住的表面形状（表面鱼）和A. mexicanus cavefish从表面鱼类祖先（12综述）演变而来。 cavefish种群已经进化了许多特性，包括眼睛的损耗，减少或色素沉着的损失，增加了味蕾和颅神经丘和改变的号码行为，如办学行为的损失，增加侵略，在喂养姿势和亢进13的变化-19。 Cavefish和表面鱼的间，和遗传作图实验已经进行识别位点和候选基因性状的洞穴1,20-26。一些候选基因已经过测试，在细胞培养1,19促进洞穴性状的职能作用，在其他物种中的21模式生物或过表达27或短暂击倒ü唱歌吗啉28 A. mexicanus。然而，这些方法有局限性。生成这些基因在A的突变体等位基因的能力mexicanus对于理解它们的功能在cavefish的演变是至关重要的。因此，A。 mexicanus是基因编辑技术的应用的理想候选有机体。
在这里，我们概括为A.基因组编辑的方法mexicanus使用TALENS。这种方法可用于评估镶嵌注射创始人鱼的表型和用于分离在感兴趣29基因稳定突变鱼线。
Protocol
Representative Results
Discussion
大踏步近年来已取得一了解性状进化的遗传基础。虽然一些性状的进化底层候选基因已被确定，它由于缺乏最进化有趣物种遗传易处理的仍然是具有挑战性的，以测试在体内这些基因。这里，我们报告中的A.基因组编辑的方法mexicanus，使用一个物种，研究洞穴动物的进化。遗传图谱研究1,21,23和候选基因方法19,40确定了一些候选基因的性状在A.洞穴形式的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由遗传，发育与细胞生物学和爱荷华州立大学的系和国立卫生研究院授予EY024941（WJ）。DR资助。杰弗里Essner提供稿件的意见。
Materials
| 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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