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。

所有的动物程序是按照美国国立卫生研究院的指导方针和机构动物护理和使用委员会在爱荷华州立大学和马里兰大学获得批准。 1. TALEN设计<ol><li>输入所需的靶序列到TALEN设计网站。 （例如: <a href="https://tale-nt.cac.cornell.edu/node/add/talen">https://tale-nt.cac.cornell.edu/node/add/talen</a> ）。输入选择间隔/复读数组的长度。 <ol><li>基因组序列拷贝到标有"序"框。 </li><li>内"提供定制间隔/ RVD长度"选项卡中选择间隔长度和数组的长度。 注:15个碱基对长度的间隔，重复15-17工作数组长度很好，使组装不太复杂。 </li></ol></li><li>选择TALEN对。围绕一个独特的限制性酶位点而设计TALEN对允许通过限制酶的基因分型消化一个PCR产物。 </li><li>设计引物扩增周围使用的网站，如:引物的30-32 TALEN目标站点的基因组区域。当用限制性酶进行基因分型，设计引物来扩增包含该限制酶位点只有一次的区域。因此建议该区域被放大和前TALEN施工，以确定存在于A.任何多态性测序要使用mexicanus实验室人口显微注射。 </li></ol> 2. TALEN大会（从TALEN包协议修改）33,34 有关其他详细信息和故障排除，请参阅该协议34。 <ol><li>备，并根据制造商的说明从TALEN试剂盒测序必要质粒。 </li><li>设立＃的反应，A和B. 1反应包括重复变量diresidues（RVDS）1-10和反应A.目的载体pFUS_A包括RVDS 11-（N-1）和第È目标向量pFUS_B在反应B（N-1），其中N是在TALEN RVDS的总数。 <ol><li>添加到每个反应:1微升含有各RVD（100毫微克/微升），目标向量（100毫微克/微升），1微升BSA限制性内切酶，1μl的牛血清白蛋白（BSA）（2毫克/毫升每种质粒的），1微升连接酶，2微升10×连接酶缓冲液和x的微升水为20微升的总体积。例如，参照表1。 注:半反应也可以用。 </li><li>将反应在热循环和运行周期:10倍（37°C / 5分钟+ 16°C / 10分钟）+ 50°C / 5分钟+ 80°C / 5分钟。 </li></ol></li><li>孵育核酸酶反应。各反应加1微升25毫摩尔ATP和1微升核酸。在37℃孵育反应1小时。 </li><li>变换反应。 <ol><li>变换2.5微升每个反应到25微升化学感受态细胞。 注:自制能力Ç厄尔可以使用。然而，随着低能力的细胞可能会导致缺乏殖民地。 <ol><li>混合2.5微升25微升化学感受态细胞的反应。在冰上孵育五分钟。孵育30秒将细胞在42℃。 </li><li>放置在冰上的管2分钟。加入125微升中超最佳肉汤代谢产物抑制（SOC）的。摇管在37℃下1小时。 </li></ol></li><li>板100微升转化细胞在LB平板上壮观霉素（50微克/毫升）中，X-gal和异丙基β-D-1-硫代半乳糖苷（IPTG）。成长O / N在37℃。 </li></ol></li><li>挑各反应2-3的白色菌落，并使用引物pCR8_F1和pCR8_R1（ 表2）由菌落PCR确认。 <ol><li>使试剂的主结构。例如，参照表3中 。 </li><li>挑取菌落并涂抹成PCR管的底部，然后把殖民地的遗体于2ml LB与壮观霉素（50微克/毫升）。将15微升主混合物与殖民地管。 </li><li>运行以下PCR程序（ 表4） </li><li>检查的PCR（运行整个卷）上通过电泳在1.5％琼脂糖凝胶。正确的克隆将具有预期大小的带，以及一涂抹和频带的梯形。为适当的涂抹的一个例子，见34,33。 </li><li>生长2毫升培养物在LB培养基O / N的正确的克隆于37℃在振荡培养箱中。 </li></ol></li><li>根据制造商的说明小量制备质粒。 </li><li>顺序检查TALEN序列以pCR8_F1和pCR8_R1。按照前述方法35。 </li><li>利用测序验证正确的克隆，建立了反应＃2（TALEN包），这将放置在A和B的载体和最终RVD的RVDS到目标向量。 <ol><li>制备用于反应2（ 表5）的组合。 注:哈LF反应都可以使用。 </li><li>发生在一个热循环反应，并运行下面的程序:37℃/ 10分钟+ 16°C / 15分钟+ 37°C / 15分钟+ 80°C / 5分钟。 </li></ol></li><li>变换反应。 <ol><li>变换2.5微升每个反应到25微升化学感受态细胞。 <ol><li>混合2.5微升25微升化学感受态细胞的反应。在冰上孵育5分钟。热休克细胞30秒，在42℃。 </li><li>放置管在冰上。加入125微升SOC的。放置在振荡培养箱管在37℃下1小时。 </li></ol></li><li>板100微升转化细胞在LB平板上的氨苄青霉素（100微克/毫升）中，X-gal和IPTG的。成长O / N，37°C。 </li></ol></li><li>挑1-3每个反应的白色菌落，并通过使用引物TAL_F1和TAL_R2（ 表2）菌落PCR检查。 <ol><li>使聚合酶mastermix，水和引物作为溶液在表3中，选择一个殖民地，它涂抹到PCR管的底部，然后把殖民地的残骸分成2毫升LB氨苄青霉素（100微克/毫升）。放置15微升溶液与集落的管。 </li><li>运行PCR程序（ 表6）。 </li><li>检查通过电泳在1.5％琼脂糖凝胶对PCR。正确的克隆将有拖尾和乐队的阶梯。为适当的涂抹的一个例子，见34,33。 </li><li>生长2毫升培养物在LB培养基O / N的正确的克隆于37℃在振荡培养箱中。 </li></ol></li><li>根据制造商的说明小量制备质粒。 </li><li>顺序检查TALEN序列以TAL_F1和TAL_R2以下先前描述的方法35。 </li></ol> 3. TALENS的转录表达<ol><li>消化序列验证模板的4微克与2微升萨克我2小时，在37℃。 </LI&gt; <li>上通过电泳在1.5％琼脂糖凝胶上运行2微升萨克消化的质粒。被正确地消化质粒将显示单一条带。 </li><li>净化后的PCR纯化试剂盒协议其余囊消化的质粒。与之前洗脱洗涤溶液洗涤两次。洗脱到的无核酸酶水30微升。 </li><li>遵循T3的mRNA生产的标准协议。 <ol><li>设置使用线性模板的0.5微克半反应（上述制备）。孵育在37℃下2小时。 </li><li>添加脱氧核糖核酸酶（包含在试剂盒）0.5微升，在37℃下孵育15分钟。 </li></ol></li><li>净化的mRNA，按照制造商的说明进行操作。洗脱到30μl的无核酸酶水。 </li><li>通过电泳跑1.5％琼脂糖凝胶来检查的mRNA。 <ol><li>清洁凝胶设备与产品，以消除RNase污染，并准备一个1.2％的凝胶。 </li><li>混合料1微升mRNA的+4＃181:L不含核酸酶的水+ 5微升乙醛酰样染料。在50℃孵育样品30分钟。离心机在运行前凝胶管短暂地置于冰上。 </li></ol></li><li>检查用定量核酸浓度的方法，该浓度并存储在等分试样的RNA在-80℃。选择的等分试样尺寸，使得RNA的不冻结/解冻一次以上。 注:500-1000纳克的浓度/通常获得NL。 （由带，而不是在凝胶上的涂抹评定），为RNA的完整性被保持具有较低浓度的RNA可以用作长。 </li></ol> 4.注入Astyanax mexicanus胚胎TALEN表达<ol><li>准备工具注入。 <ol><li>通过在鱼水注入1.2％琼脂糖（水用碳酸氢钠和海盐至pH 7.4，电导率700μS空调）到一个培养皿倾注射板。将模具（与预测塑料片，使鱼井鸡蛋）的菜里面。除去当琼脂糖硬化的模具和储存在4℃。有关模具详见36。 </li><li>拉使用根据制造商的说明的针拔出器的注射针。 注意:适当的针的长度是注射重要。针太长会打针太灵活。拉针的协议将用来拉针设备的不同而不同。合适的针的一个例子示于图1。对于我们的设备，我们拉是5-6厘米长针，以及破碎时在约0.011毫米尖端的外径。然而，针头应校准（步骤4.3.4），以确定开口宽度。拉针的示例程序可以在表7中找到。 </li><li>通过打破吸管使开口足够大的鸡蛋通过准备胚胎移植玻璃吸管。火焰破月底到它不再尖锐。 注意:使用玻璃吸管和玻璃碗重要的是与A.时mexicanus卵子和胚胎，因为他们是粘，将坚持以塑料。 </li></ol></li><li>收集1-细胞阶段的鸡蛋。 <ol><li> A.品种mexicanus以下标准协议37。 注:例如，如果鱼保持在14光:10暗周期和和ZT14使用Zeitgerber时间（ZT）与ZT0作为灯的灯关，我们的ZT15和ZT19的表面鱼卵。确切的产卵时间必须为每个单独的实验室来确定。 </li><li>通过交配之前过度喂食的鱼3-4天，并把鱼放入清水催产。提高温度2°F。注:我们最初的水温为74左右°F。 </li><li>表面收集鱼卵在黑暗中，检查每15分钟在1细胞阶段获得鸡蛋。数百蛋可以从一个单一的一对表面的鱼获得。 </li><li>搜集鸡蛋在玻璃碗防止粘到塑料表面和排序，以在1-细胞期胚胎注射前通过在显微镜下观察卵和收集卵是单个细胞分离。保持蛋在新鲜的系统中的水（水箱，其中成鱼被容纳，这已为pH和电导率处理的）。 </li></ol></li><li>注射TALEN表达。 <ol><li>注入不同量的总mRNA（在一对等量各TALEN的），以确定注射的最佳浓度为毒性和效率由TALEN对而异。通过注入总mRNA的浓度是400-800皮克开始。稀释并结合基因到所需的浓度为1.5注射NL。 </li><li>负载稀释的mRNA进针的背面和针附着到微注射器。 </li><li>使用镊子断针。 </li><li>校准针。例如，喷射10次，并收集在一个微毛细管所得降。 10×100一次性1.081; L，32毫米微毛细管，下降应填写微毛细管至0.5毫米，1.5升/注射1次。根据需要调整注射时间和压力。 </li><li>针插入的单电池和注入的mRNA。直接注入​​基因进入细胞内，没入蛋黄。 </li><li>收集在玻璃碗注射胚胎。胚胎保持在23-25​​℃。除去死胚定期注射后的头几天（即变得浑浊和不规则形胚）。从控制（未注射）和注射板死亡，畸形胚胎创纪录的数字。 注:增加mRNA的浓度会导致增加毒性和胚胎畸形/死亡。因此，相对于毒性效率必须平衡，以确定mRNA的最佳浓度注入。 </li></ol></li></ol> 5.表型方正鱼和评估TALEN效率<ol><li>根据机构的动物祭祀协议胚胎。 注意:我们安乐死胚胎通过快速冷却冰。 </li><li>收集胚胎成用移液0.8微升PCR联管。 注:基因分型可在单个胚胎或胚胎的池来进行。 </li><li>提取DNA。 <ol><li>地方胚胎入100微升50氢氧毫氢氧化钠（NaOH）和孵育在95℃下进行30分钟，然后冷却至4℃。 </li><li>添加的1M的Tris-HCl pH为8的1/10体积（10微升）。 </li></ol></li><li>使用在步骤1.3所设计的引物（ 见表8和9样品的协议）的区域执行PCR。个别胚1微升的DNA是足够的PCR反应。 </li><li>消化用合适的限制性酶将所得的PCR产物，并通过电泳运行1.5％琼脂糖凝胶。 <ol><li>例如，对于在注射的胚胎基因分型的眼皮肤白化病2（OCA2）位点用限制酶BSR我通过加入0消化PCR产物0.5微升BSR我直接的12.5微升完成PCR反应，在65℃下2小时温育。运行未消化和在凝胶中消化产物。限制性内切酶抗性带（ 即 ，不易消化频带）表明TALEN诱发突变都存在（ 图2）。 </li></ol></li><li> （可选的）通过使用凝胶分析工具如先前29描述的来计算每个频带的强度值在斐济38分析的凝胶的图像确定未切割产物的百分比计算的百分比的突变率。 </li><li>由TA克隆凝胶纯化限制酶抗性突变体带和测序克隆确定突变等位基因的序列。 <ol><li>凝胶纯化限制按照制造商的说明书酶抗性突变体带。 TA克隆带按照制造商的说明进行操作。挑选殖民地和在摇动生长在1.5ml LB O / N在37℃孵化器。 </li><li>小量制备培养物按照制造商的说明进行操作。发送该DNA测序。 </li><li>使用程序如猿，对准突变序列与野生型序列。 <ol><li>复制并粘贴两个序列成APE文件。选择"工具"菜单中的"对齐两个序列"的​​工具。 </li><li>指定使用下拉菜单两个DNA序列。 注意:克隆的序列的反向互补可能需要根据在PCR进入克隆载体的方向被使用。如果序列不对齐，重复步骤检查在"对齐DNA"中的"启-Com的"框。 </li></ol></li></ol></li><li>评价创始人鱼在适当阶段的表型和使用用于预期表型29适当的方法（例如， 图3）。表型的方法，将基于使用protoc用于基因预期的表型由研究人员的目标醇。 </li></ol> 6.屏幕种系传递注:A. mexicanus在4-8个月达到性成熟。 <ol><li>交叉性成熟创始人鱼类与野生型鱼。 </li><li>屏幕胚或利用在步骤5.1-5.7方法成鱼（ 图4）。对于成年鱼，一块尾巴可按照麻醉被裁剪。 <ol><li>通过在三卡因（3-氨基苯甲酸乙酯）的溶液中浸没鱼减少翅片削波期间的应力麻醉鱼。继鳍剪裁，让鱼在淡水中恢复。鱼快速恢复;观察直至病愈（通常是游泳）。 </li></ol></li></ol>

The authors have nothing to disclose.

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?...

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。

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thermocycler</td><td></td><td></td><td></td></tr><tr><td>Injection station</td><td></td><td></td><td></td></tr><tr><td>Gel apparatus</td><td></td><td></td><td></td></tr><tr><td>Needle puller</td><td></td><td></td><td></td></tr><tr><td>Nanodrop</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Note: As far as we know, supplies from different companies can be used unless otherwise indicated</td><td></td><td></td><td></td></tr><tr><td>Golden Gate TALEN and TAL Effector Kit 2.0</td><td>Addgene</td><td>Kit #1000000024</td><td></td></tr><tr><td>Fisher BioReagents LB Agar, Miller (Granulated)</td><td>Fisher</td><td>BP9724-500</td><td></td></tr><tr><td>Fisher BioReagents Microbiology Media: LB Broth, Miller</td><td>Fisher</td><td>BP1426-500</td><td></td></tr><tr><td>Teknova TET-15 in 50% EtOH</td><td>Teknova (ordered through Fisher)</td><td>50-843-314</td><td></td></tr><tr><td>Spectinomycin Dihydrochloride, Fisher BioReagents</td><td>Fisher</td><td>BP2957-1</td><td></td></tr><tr><td>Super Ampicillin (1,000x solution)</td><td>DNA Technologies</td><td>6060-1</td><td></td></tr><tr><td>ThermoScientific X-Gal Solution, ready-to-use</td><td>Thermo Sci Fermentas Inc (Ordered through Fisher)</td><td>FERR0941</td><td></td></tr><tr><td>IPTG, Fisher BioReagents</td><td>Fisher</td><td>BP1620-1</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher</td><td>08-757-13</td><td></td></tr><tr><td>&lt;em&gt;Bsa&lt;/em&gt;I</td><td>New England Biolabs (ordered through Fisher)</td><td>50-812-203</td><td>Use BsaI, not BsaI-HF (as described in the Golden Gate TALEN and TAL Effector Kit protocol)</td></tr><tr><td>BSA</td><td>New England Biolabs</td><td>provided with restriction enzymes</td><td></td></tr><tr><td>10x T4 ligase buffer</td><td>Promega (ordered through Fisher)</td><td>PR-C1263</td><td></td></tr><tr><td>GoTaq Green Master mix</td><td>Promega (ordered through Fisher)</td><td>PRM7123</td><td>Other Taq can be used, but the reaction should be adjusted accordingly</td></tr><tr><td>Quick ligation kit</td><td>New England Biolabs (ordered through Fisher)</td><td>50-811-728</td><td>We use Quick Ligase for all TALEN assembly reactions</td></tr><tr><td>One Shot TOP10 Chemically Competent E.coli</td><td>Invitrogen</td><td>C4040-06</td><td>Other chemically competent cells or homemade competent cells can be used</td></tr><tr><td>&lt;em&gt;Esp&lt;/em&gt; 3I</td><td>Thermo Sci Fermentas Inc (Ordered through Fisher)</td><td>FERER0451</td><td></td></tr><tr><td>Plasmid-Safe ATP-dependent DNase</td><td>Epicentre (Ordered through Fisher)</td><td>NC9046399</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>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></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>GeneMate LE Quick Dissolve Agaraose</td><td>BioExpress</td><td>E-3119-125</td><td></td></tr><tr><td>&lt;em&gt;Sac&lt;/em&gt; I</td><td>Promega (Ordered through Fisher)</td><td>PR-R6061</td><td></td></tr><tr><td>mMESSAGE mMACHINE T3 Transcription kit</td><td>Ambion</td><td>AM1348M</td><td></td></tr><tr><td>Rneasy MinElute Cleanup Kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>NorthernMax-Gly Sample Loading Dye&amp;nbsp;</td><td>Ambion (ordered through Fisher)</td><td>AM8551</td><td></td></tr><tr><td>Eliminase</td><td>Decon (ordered through Fisher)</td><td>04-355-32</td><td></td></tr><tr><td>Fisherbrand Disposable Soda-Lime Glass Pasteur Pipets</td><td>Fisher</td><td>13-678-6B</td><td></td></tr><tr><td>Standard Glass Capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td></td></tr><tr><td>Microcaps</td><td>Drummond Scientific Company</td><td>1-000-0010</td><td></td></tr><tr><td>Eppendorf Femtotips Microloader Tips for Femtojet Microinjector</td><td>Eppendorf (ordered through Fisher)</td><td>E5242956003</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fisher</td><td>S318-500</td><td></td></tr><tr><td>Tris base</td><td>Fisher</td><td>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对注射导致的FokⅠ域的二聚的RVDS到特定的DNA核苷酸的结合，因此，产生双链断裂39可通过非同源末端连接（NHEJ）进行修复。 NHEJ常常引入导致在插入或缺失（插入缺失）错误。插入缺失可以通过扩增包围TALEN目标位点的区域和消化与TALEN间隔区之内的切割的限制性酶将所得的扩增子进行识别。没有一个插入缺失的等位基因，而含有等位基因改变限制性内?...

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

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）。

基因组编辑在<em&gt; Astyanax mexicanus</em&gt;使用转录因子样效应核酸酶（TALENS）

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

genome-editing-astyanax-mexicanus-using-transcription-activator-like

Research

JoVE Journal

Biology

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

8.2K 次观看.  Iowa State University. 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）。 

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

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

科研

遗传学

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

发育生物学

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

提高墨西哥龙舌兰对幼虫后表型和全体免疫组织化学的分析

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

墨西哥鱼基因功能的操纵

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

通过CAS9蛋白-gRNA复合物电穿孔直接基因敲除Axolotl脊髓神经干细胞

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

阿斯蒂亚纳克斯胚胎的全原位杂交

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

小鼠基因组工程使用Designer核酸酶

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

基因组编辑在

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