感谢上海托洛生物科技在开发C-Brick标准方面的技术援助。这项工作得到了中国科学院战略重点研究计划（批准号:XDB19040200）的资助。

1.制备crRNA <ol><li> 制备crRNA模板 <ol><li>将无RNase的水中的单个寡核苷酸（ 表1 ）重新悬浮至10μM的浓度。 </li><li>加入22.5μL顶链寡核苷酸（ 表1中 T7-F），22.5μL底链寡核苷酸（ 表1 ）和5μL10x退火缓冲液至0.2mL PCR管。确保总体积为50μL。 注意: 表1中显示了六种不同的底链寡核苷酸，每种寡核苷酸应与顶链T7-F单独配对。 表1中的小写字母表示要转录为crRNA指导序列的序列。可以使用10x Taq PCR缓冲液代替10x退火缓冲液。 </li><li>将PCR管放入热循环仪中。 </li><li>执行退火程序:95℃下初始变性5分钟，然后冷却从95-20℃，热循环仪每分钟降低1℃。 注意:立即使用样品进行步骤1.2.1。 </li></ol></li><li> 转录和纯化的crRNA <ol><li>加入38μL无RNase的水，8μL来自步骤1.1.4的模板，20μL5x T7转录缓冲液，20μLNTP混合物（10μM），4μL重组RNA酶抑制剂（RRI）和10μl μLT7 RNA聚合酶到1.5 mL微量离心管中。确保总体积为100μL。 </li><li>将微量离心管放入37°C水浴过夜（约16 h）。 </li><li>使用RNA清除和浓缩试剂盒来纯化转录的RNA;遵循制造商的协议。 </li><li>用UV-Vis分光光度计定量RNA，并将RNA稀释至10μM浓度。立即使用样品或将其储存在-80°C。 注意:crRNA序列s列于表2 。 </li></ol></li></ol> 2. C-Brick零件的构造（ 即将生物零件插入C砖标准矢量） 注意:此步骤有三个子步骤。对于短生物部分（ 如启动子和终止子），最好使用直接PCR方法将生物部位插入C-Brick标准载体4 。整个矢量序列显示在补充数据中，前缀和后缀序列如图1所示 （下面的步骤2.1）。对于可以通过PCR容易获得的生物部位（ 例如，使用基因组DNA，质粒或从头合成的DNA序列的模板），最好使用无缝组装方法将生物部位插入C-Brick标准载体（下面的步骤2.2）。对于从BioBrick和BglBrick标准获得的那些部件，限制酶介导的消化和T4 DNA连接酶介导的连接可用于将生物部位插入C-Brick标准载体（下文的步骤2.3）。 <ol><li> 通过直接PCR扩增构建 <ol><li>设计和订购PCR扩增的寡核苷酸。 注意:寡核苷酸的5&#39;末端含有DNA部分的序列，寡核苷酸的3&#39;末端含有载体序列（ 即，前向3&#39;末端的"GGATCCACTAGTCTCTAGCTCG"和"GGATCCTTTCTCCTCTTTCTAG"在反向链中）。可以在寡核苷酸的5&#39;端设计无突出端或〜20-bp突出端。具体的例子可以在以前的研究中找到。 </li><li>将超纯水中的单个寡核苷酸重新悬浮至10μM的浓度。 </li><li>在0.2 mL PCR管中，在冰上设置PCR反应:加入18μL超纯水，25μL2x PCR缓冲液，1μLdNTP（10μM），2.5μL来自步骤2.1.2的正向和反向引物（10μM），0.5μL作为模板的C-Brick标准载体（50ng）和0.5μL的高保真DNA聚合酶。确保总体积为50μL。 </li><li>将管直接放入热循环仪中，启动热循环仪程序。立即使用样品或将其冷冻保存在-20°C。 </li><li>热循环程序:98℃一个循环3分钟; 98℃30秒，55℃20秒，72℃2秒;在72℃下循环10分钟。将样品保持在16°C。 </li><li>将9μL步骤2.1.4的混合物加入到1.5 mL微量离心管中。 </li><li>加入1μLDpnI，并在37°C水浴中孵育1 h。 注意:如果引物没有重叠序列，加入0.5μLT4多核苷酸激酶（PNK）和0.5μLT4 DNA连接酶，并孵育22°C水浴1 h。否则，如果引物含有〜20nt的重叠序列，则将DpnI处理的产物直接转化到大肠杆菌 （DH10B）感受态细胞中（步骤2.4）。线性化PCR产物将通过宿主中的重组系统进行环化。 </li><li>立即使用样品或将其储存在-20°C。 </li></ol></li><li> 通过无缝组装施工 <ol><li>设计和订购PCR扩增的寡核苷酸。 注意:寡核苷酸的3&#39;末端含有DNA部分的末端序列，寡核苷酸的5&#39;末端包含线性化C-Brick标准载体序列的末端（ 即 "CTAGAAAGAGGAGAAAGGATCC"为5&#39;前端的末端和反义链5&#39;端的"CGAGCTAGAGACTAGTGGATCC"）。具体的例子可以在以前的研究中找到。 </li><li>重新挂起个人在超纯水中的寡核苷酸浓度为10μM。 </li><li>在0.2 mL PCR管中，在冰上设置PCR反应:加入18μL超纯水，25μL2x PCR缓冲液，1μLdNTP（10μM），2.5μL正向和反向引物（10μM ），0.5μL的模板（20-500ng）和0.5μL的高保真DNA聚合酶。确保总体积为50μL。 </li><li>将管直接放入热循环仪中，运行热循环仪程序。立即使用样品或将其冷冻保存在-20°C。 注意:将热循环仪程序设置为:在98℃下一个循环3分钟;在98℃下30秒，55℃20秒（根据引物的退火温度）和在72℃下1分钟（根据生物部分的长度）30个循环;在72℃下循环10分钟。将样品保持在16°C。 </li><li>使用PCR清理系统纯化PCR产物工具箱遵循制造商的协议。 </li><li>用BamHI消化C-Brick标准载体:向微量离心管中加入34μL超纯水，10μL质粒（1μg），5μL10x缓冲液和1μLBamHI。在37℃下在水浴中孵育2小时。 </li><li>使用凝胶清理系统套件净化上述产品;遵循制造商的协议。 </li><li>加入2μL来自步骤2.2.7的线性化载体（50ng），5μL来自步骤2.2.4的片段（200ng），2μL5x无缝组装缓冲液和1μL无缝组装酶的无缝组装酶至1.5 mL微量离心管。确保总体积为10μL。 </li><li>将其放在37°C水浴中30分钟。立即使用样品或将其冷冻保存在-20°C。 </li></ol></li><li> 通过限制酶介导的消化和T4 DNA连接酶介导的连接进行构建。 注意:对于从BioBrick（步骤2.3.4-2.3.7）和BglBrick（步骤2.3.1-2.3.3）标准品获得的那些部分，可以使用限制酶介导的消化和T4 DNA连接酶介导的连接来插入生物部位成为C-Brick标准载体。 <ol><li>用BamHI和BglII对BglBrick标准品进行消化:向微量离心管中加入34μL超纯水，10μL质粒（2μg），5μL10x缓冲液3，0.5μLBamHI和0.5μLBglII。在37°C水浴中孵育2小时。 </li><li>进行1％琼脂糖凝胶电泳，并使用凝胶清理系统试剂盒纯化该片段。 </li><li>将片段和C-Brick标准载体连接:将2μL来自步骤2.1.12的线性化载体（50ng）和6μL来自步骤2.3.3的片段（200ng）加入到1.5mL微量离心管中。加入1μL10 x T4 DNA连接酶缓冲液和1μLT4 DNA连接酶。在22°C孵育2小时。立即使用样品或冻结e并将其储存在-20°C。 </li><li>用XbaI和SpeI对BioBrick标准件进行摘录:向微量离心管中加入34μL超纯水，10μL质粒（2μg），5μL10x缓冲液，0.5μLXbaI和0.5μLSpeI。在37°C水浴中孵育2小时。 </li><li>用XbaI和SpeI对C-Brick标准载体进行消化:向微量离心管中加入34μL超纯水，10μL质粒（1μg），5μL10x缓冲液，0.5μLXbaI和0.5μLSpeI 。在37°C水浴中孵育2小时。 </li><li>进行1％琼脂糖凝胶电泳，并用凝胶清洗系统试剂盒纯化步骤2.3.4的片段和来自步骤2.3.5的线性化载体;遵循制造商的协议。 </li><li>将片段和C-Brick载体连接:将2μL来自步骤2.3.5的线性化载体（50ng）和6μL来自步骤2.3.4的片段（200ng）加入到1.5mL微量离心管中。广告d 1μL10×T4 DNA连接酶缓冲液和1μLT4 DNA连接酶。在22°C孵育2小时。立即使用样品或将其冷冻保存在-20°C。 </li></ol></li><li>加入10μL产品，步骤2.1.8,2.2.9,2.3.3或2.3.7进行大肠杆菌感受态细胞化学转化 （DH10B）。 </li><li>将几个克隆接种到5-mL液体Luria-Bertani（LB）管中，并在37℃下在振荡器（220rpm）上孵育过夜。 </li><li>使用质粒制备试剂盒提取质粒;遵循制造商的协议。 </li><li>通过Sanger测序鉴定正确的克隆11 。 </li></ol> 3. C砖装配（ 图2 ） <ol><li> 用Cpf1消化C-Brick载体 <ol><li>加入22.5μL不含RNase的水，4μL10x Cpf1缓冲液，10μL来自步骤2.6（1μg）的质粒，0.5μLRRI和1μl＃181;将Cpf1（5μM）的L加到1.5-mL微量离心管中。 注意:Cpf1可以在以前的研究中按照协议在商业上购买或纯化4 。 </li><li>为了将外源DNA片段插入到T1和T2位点，加入1μL的crRNA-T2（10μM），并在37℃的水浴中孵育30分钟。 将 ddl crRNA-T1（10μM）和Cpf1（5μM）另外30分钟。 注意:另外，为了将外源DNA片段插入T3和T4位点，加入1μL的crRNA-T3（10μM），并在37℃的水浴中孵育30分钟。再加入1μL的crRNA-T4（10μM）30分钟。 </li><li>加入1μL热敏碱性磷酸酶，并将管在37°C水浴中孵育另外1 h。 </li><li>进行琼脂糖凝胶电泳，并使用凝胶清理系统试剂盒纯化载体。立即使用样品或将其冷冻保存在-20°C。 </li></ol></LI&gt; <li> 用Cpf1消化DNA片段 <ol><li>向1.5 mL微量离心管中加入21.5μL无RNase的水，4μL10x Cpf1缓冲液，10μL质粒，步骤2.6（2μg），0.5μLRRI和2μLCpf1（5μM）。 </li><li>要将外来DNA片段插入T1和T2位点，加入1μL的crRNA-T1（10μM）和1μL的crRNA-T3（10μM），并在37℃的水浴中孵育2小时。 注意:或者，加入1μL的crRNA-T2（10μM）和1μL的crRNA-T4（10μM），并在37℃的水浴中孵育2小时。 </li><li>运行琼脂糖凝胶电泳，并使用凝胶清理系统套件纯化该片段。立即使用样品或将其冷冻保存在-20°C。 </li></ol></li><li> C砖组装和进一步验证 <ol><li>连接外来DNA片段和C-Brick载体:加入2μL线性化载体（50ng）和6μL来自步骤3.2.3的DNA片段（200ng）至1.5mL微量离心管。加入1μL10×T4 DNA连接酶缓冲液和1μLT4 DNA连接酶。在22°C孵育2小时。立即使用样品或将其冷冻保存在-20°C。 </li><li>加入10μL连接产物（步骤3.3.1）进行大肠杆菌 （DH10B）感受态细胞的化学转化。 </li><li>将几个克隆添加到5 mL液体LB管中，并在振荡器上以220 rpm和37°C孵育过夜。 </li><li>使用质粒制备试剂盒提取质粒;遵循制造商的协议。 </li><li>通过Sanger测序鉴定正确的克隆11 。 </li></ol></li><li>执行新一轮C-Brick装配。步骤3.3.4中的正确克隆可以用作新的C-Brick向量或DNA部分，用于进一步迭代C-砖装配，遵循步骤3.1-3.3中相同的协议。 注意:T2&#39;和T3&#39;它被设计为T2和T3站点的备份（ 图1a ）。对于从步骤2.3.7获得的质粒，只有T2&#39;和T3&#39;可用于C-Brick标准组装。 </li></ol>

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作者没有什么可以披露的。

该协议描述了DNA组装标准C-Brick的程序。该协议中最重要的一步是C-Brick标准向量的线性化;载体的不完全切割可能严重影响成功率。另外，虽然Cpf1主要以"18-23"切割模式切割目标DNA序列，但也检测到两个碱基附近的不准确的切割，这可能在DNA组装后引起少量突变。因此，需要Sanger测序来验证组装的构建体。 C-Brick组件的效率低于以前研究中已经描述的传统限制酶和连接方法<sup cl...

DNA生物部件的标准化对合成生物学的发展至关重要1 。 DNA组装过程的发展可以取代特设实验设计，并消除在遗传组分装配到更大系统中时出现的许多意想不到的结果。 BioBrick标准（BBF RFC 10）是最早提出的DNA装配标准之一。它使用前缀序列（含有EcoRI和XbaI切割位点）和后缀序列（含SpeI和PstI切割位点） 2,3 。由于XbaI和SpeI具有互补性，所以用XbaI和SpeI切割的BioBrick DNA部件可以连接在一起，生成一个新的BioBrick，用于进一步的迭代装配。 已经使用BioBrick标准鉴定了一些缺陷4 。例如，它产生8-bp的瘢痕在DNA部分之间，其不允许构建融合蛋白。此外，上述四种类型的6-bp限制性位点必须从DNA部分中去除，这是非常不方便的。 BglBrick标准是为了解决第一个问题而建立的。它产生6-bp"GGATCT"瘢痕，产生Gly-Ser并允许多个蛋白质或蛋白质结构域的融合。 iBrick被开发来处理第二个问题6 。它使用识别长DNA序列的归巢内切核酸酶（HE）。由于HE识别位点在天然DNA序列中很少存在，所以iBrick标准可用于直接构建iBrick部分而不修改其DNA序列。然而，iBrick标准在DNA部分之间留下了21bp的瘢痕，这可能是其不受欢迎的原因。 近年来，群集定期交织的短回文重复序列（CRISPR）系统发展迅速7,8 。在CRISPR相关（Cas）蛋白中，来自化脓性链球菌的 Cas9核酸内切酶现在被广泛使用。它主要引入具有钝端的双链DNA断裂（DSB） 9 。 2015年，张和同事首次以Cpf1（来自Prevotella和Francisella 1的CRISPR）为特征。属于2类V型CRISPR-Cas系统，是CRISRP RNA（crRNA）引导型核酸内切酶10 。与Cas9不同，Cpf1引入了具有4或5 nt 5&#39;突出端的DSB 10 。基于此特征，Cpf1用于开发DNA组装标准C-Brick 4 。在C-Brick标准载体上，四个具有前缀T1 / T2和后缀T3 / T4的Cpf1靶位点位于生物部位的侧面;这与BioBrick标准相似。随着T2和T3位点的切割p导致互补的粘性末端，可以在部件之间产生"GGATCC"疤痕的同时执行DNA部件的迭代装配。值得注意的是，C-Brick标准有两个主要优点:识别长目标序列并留下短疤痕。由C-Brick产生的6bp"GGATCC"瘢痕编码Gly-Ser，其允许构建融合蛋白。此外，C-Brick标准也与BglBrick和BioBrick标准部分兼容。

<table><tbody><tr><td>Comercial Oligonucleotide</td><td>Sangon Biotech</td><td></td><td></td></tr><tr><td>10x Taq PCR Buffer</td><td>Transgen</td><td>#J40928</td><td></td></tr><tr><td>Ultra Pure Distilled Water</td><td>Invitrogen</td><td>10977-015</td><td></td></tr><tr><td>5x RNA Transcription Buffer</td><td>Thermo Scientific</td><td>K0441</td><td></td></tr><tr><td>T7 RNA polymerase</td><td>Thermo Scientific</td><td>#EP0111</td><td></td></tr><tr><td>NTP mixture</td><td>Sangon</td><td>#ND0056</td><td></td></tr><tr><td>RRI (Recombinant RNase Inhibitor)</td><td>Takara</td><td>2313A</td><td></td></tr><tr><td>RNA Clean &amp;amp; Concentrator-5</td><td>Zymo Research</td><td>R1015</td><td></td></tr><tr><td>UV-Vis Spectrometer</td><td>Thermo Scientific</td><td>Nano-Drop 2000c</td><td></td></tr><tr><td>2x Phanta Max Buffer</td><td>Vazyme</td><td>PB505</td><td>PCR buffer</td></tr><tr><td>dNTPs</td><td>Transgen</td><td>AD101</td><td></td></tr><tr><td>Phanta Max Super-Fidelity DNA Polymerase</td><td>Vazyme</td><td>P505-d1</td><td></td></tr><tr><td>Ezmax for One-step Cloning</td><td>Tolobio</td><td>24303-1</td><td>seamless assembly kit</td></tr><tr><td>5x Buffer for Ezmax One-step Cloning</td><td>Tolobio</td><td>32006</td><td></td></tr><tr><td>BamHI</td><td>NEB</td><td>#R0136L</td><td></td></tr><tr><td>BamHI-HF</td><td>NEB</td><td>#R3136L</td><td></td></tr><tr><td>BglII&amp;nbsp;</td><td>NEB</td><td>#R0144L</td><td></td></tr><tr><td>XbaI</td><td>NEB</td><td>#R0145L</td><td></td></tr><tr><td>SpeI</td><td>NEB</td><td>#R3133L</td><td></td></tr><tr><td>10x Buffer 3</td><td>NEB</td><td>#B7003S</td><td></td></tr><tr><td>10x CutSmart Buffer</td><td>NEB</td><td>B7204S</td><td></td></tr><tr><td>10x T4 DNA ligase Buffer</td><td>Tolobio</td><td>32002</td><td></td></tr><tr><td>T4 PNK</td><td>Tolobio</td><td>32206</td><td></td></tr><tr><td>T4 DNA ligase</td><td>Tolobio</td><td>32210</td><td></td></tr><tr><td>DpnI</td><td>NEB</td><td>#R01762</td><td></td></tr><tr><td>SV Gel and PCR clean-up system</td><td>Promega</td><td>A9282</td><td></td></tr><tr><td>Plasmid Mini Kit I</td><td>Omega</td><td>D6943-02</td><td>plasmid preparation kit</td></tr><tr><td>thermosensitive alkaline phosphatase&amp;nbsp;</td><td>Thermo Scientific</td><td>#EF0651</td><td>FastAP</td></tr><tr><td>10x Cpf1 buffer</td><td>Tolobio</td><td>32008</td><td></td></tr><tr><td>Cpf1</td><td>Tolobio</td><td>32105</td><td>FnCpf1</td></tr><tr><td>thermocycler</td><td>Applied Biosystems</td><td>veriti 96 well</td><td></td></tr><tr><td>C-Brick standard vector</td><td>Tolobio</td><td>98101</td><td></td></tr><tr><td>E. coli [DH10B]</td><td>Invitrogen</td><td>18297010</td><td></td></tr><tr><td>Luria-Bertani media (tryptone)</td><td>Oxoid</td><td>LP0042</td><td></td></tr><tr><td>Luria-Bertani media (yeast extract)</td><td>Oxoid</td><td>LP0021</td><td></td></tr><tr><td>Luria-Bertani media (NaCl)</td><td>Sangon Biotech</td><td>B126BA0007</td><td></td></tr></tbody></table>

protocols for c-brick dna standard assembly using cpf1

CRISPR相关蛋白Cpf1在CRISPR RNA（crRNA）的指导下切割双链DNA，产生粘性末端。由于这个特点，Cpf1已被用于建立称为C-Brick的DNA装配标准，其具有长识别位点和短瘢痕的优点。在标准C-Brick载体上，有四个Cpf1识别位点 - 前缀（T1和T2位点）和后缀（T3和T4位点） - 侧翼生物DNA部分。 T2和T3位点的切割产生互补的粘性末端，这允许用T2和T3位点组装DNA部分。同时，组装后部件之间产生短暂的"GGATCC"疤痕。由于新形成的质粒再次含有四个Cpf1切割位点，所以该方法允许DNA部分的迭代装配，这与BioBrick和BglBrick标准相似。概述使用C-Brick标准来组装DNA部分的程序在这里描述。 C-Brick标准可以被科学家，研究生和本科生甚至业余爱好者广泛使用。

<html>该协议证明了三种生色蛋白盒（cjBlue（BBa_K592011），eforRed（BBa_K592012）和amilGFP（BBa_K592010））的组装。首先，将上述三种基因和终止子的编码序列分别克隆到C-Brick标准载体中。将短DNA部分，启动子和终止子通过PCR扩增引入到C-Brick载体中，使用在5&#39;末端含有短DNA部分的引物。之后是自连接方法。首先从头合成三种染色体编码序列，然后通过无缝组装克隆到C-Bric...

Watch this Scientific Journal Video about Protocols for C-Brick DNA Standard Assembly Using Cpf1 at JoVE.com

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR相关蛋白Cpf1可以由专门设计的CRISPR RNA（crRNA）引导，以在所需位点切割双链DNA，产生粘性末端。基于此特点，建立了DNA组装标准（C-Brick），并详细介绍了其使用方法。

使用Cpf1的C-Brick DNA标准装配的方案

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

protocols-for-c-brick-dna-standard-assembly-using-cpf1

Research

JoVE Journal

Genetics

8.2K 次观看.  Chinese Academy of Sciences.  CRISPR相关蛋白Cpf1可以由专门设计的CRISPR RNA（crRNA）引导，以在所需位点切割双链DNA，产生粘性末端。基于此特点，建立了DNA组装标准（C-Brick），并详细介绍了其使用方法。 

视频: Protocols for C-Brick DNA Standard Assembly Using Cpf1

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

一个工具包，使水环境中的烃类转化

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

科研

生物工程

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

A Customizable Protocol for String Assembly gRNA Cloning STAgR

Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

字符串装配 grna 克隆 (stacr) 的可自定义协议

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic ...

遗传学

Capturing Chromosome Conformation Across Length Scales

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population.
A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.

Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.

跨长度尺度捕获染色体构象

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) ...

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

由小圆形 DNA 分子构建的稳定 DNA 分子、一维和二维纳米结构

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...

化学

用于本科研究和教学的染色体构象捕获 （3C）

Getting an A with the 3Cs: Chromosome Conformation Capture for Undergraduates

Chromosome conformation capture (3C) is a powerful tool that has spawned a family of similar techniques (e.g., Hi-C, 4C, and 5C, referred to here as 3C techniques) that provide detailed information of the three-dimensional organization of chromatin. The 3C techniques have been used in a wide range of studies, from monitoring the changes in chromatin organization in cancer cells to identifying enhancer contacts made with gene promoters. While many of the studies using these techniques are asking big genome-wide questions with intricate sample types (i.e., single-cell analysis), what is often lost is that the 3C techniques are grounded in basic molecular biology methods that are applicable to a broad range of studies. By addressing tightly focused questions of chromatin organization, this cutting-edge technique can be used to enhance the undergraduate research and teaching lab experience. This paper presents a 3C protocol and provides adaptations and points of emphasis for implementation at primarily undergraduate institutions in undergraduate research and teaching experiences.

作者聚焦：用 3C 获得 A：本科生的染色体构象捕获  

Here, we present an adaption of the chromosome conformation capture (3C) technique in detail with an emphasis on undergraduate involvement and learning.

获得 3C 的 A：本科生的染色体构象捕获

Chromosome conformation capture (3C) is a powerful tool that has spawned a family of similar techniques (e.g., Hi-C, 4C, and 5C, referred to here as 3C ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

可重构 DNA 手风琴架的设计与合成

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

使用模块化金门克隆的多基因结构快速组装

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

重组工程同源重组构建<em&gt;果蝇</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

使用Cpf1的C-Brick DNA标准装配的方案

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