Name: gene digital circuits based on crispr-cas systems and anti-crispr proteins
Uploaded: 2022-10-18T15:00:00
Description: Watch this Scientific Journal Video about Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins at JoVE.com

我们要感谢TJU合成生物学实验室-SPST的所有学生的帮助，以及李智和张向阳在FACS实验中的帮助。

1. sgRNA/crRNA表达盒的设计与构建
注意：有两种sgRNA / crRNA表达盒：一种称为SNR5210-由RNA聚合酶III依赖性SNR52启动子，sgRNA / crRNA序列和SUP4终止子组成;另一个缩写为RGR11由RNA聚合酶II依赖性ADH1启动子，RGR（核酶引导RNA-核酶）结构组成，其中包含两个核酶（锤头核酶-HH和肝炎δ病毒核酶-HDV）和介于两者之间的sgRNA/crRNA序列?...

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该协议显示了合成基因数字电路可能的完整工作流程，遵循“设计-构建-测试-学习”（DBTL）生物工程周期，涉及干实验室和湿实验室实验。在这里，我们专注于CRISPR-Cas系统，主要是dSpCas9，denAsCas12a，dLbCas12a和相应的抗CRISPR蛋白，通过在 酿酒酵母 中设计和构建小转录网络。其中一些模仿布尔门，布尔门是数字电路的基本组件。这里描述的所有回路都允许我们描述酿 酒酵母中CRISPR相?...

2011年，研究人员提出了一种计算方法，并开发了相应的软件，用于数字合成基因电路的自动设计1。用户必须指定输入数量（三个或四个）并填写电路真值表;这提供了使用电子技术推导电路结构的所有必要信息。真值表通过 Karnaugh 映射方法2 转换为两个布尔公式。每个布尔公式都由描述电路输入（部分）之间的逻辑运算（和或乘法）及其否定（文字）的子句组成。子句依次相加（OR）或乘法（AND）以计算电路输出。每个电路都可以根据其两个相应的公式中的任何一个来实现：一个以POS（总和乘积）形式编写，另一个以SOP（产品总和）表示形式编写。前者由包含文字逻辑和的子句（即布尔门）的乘法组成。相比之下，后者是文字相乘的子句的总和。
电路可以在面包板上通过将不同的门物理连接在一起来实现。电流允许门之间交换信号，从而计算输出。
在生物学中，情况更为复杂。布尔门可以实现为转录单元（TU;即真核细胞内的序列“启动子编码区域终止子”），其中转录或翻译（或两者）受到调节。因此，至少有两种分子建立了生物线路：转录因子蛋白和非编码、反义RNA。
基因数字电路被组织成两层或三层门，即：1）输入层，由YES（缓冲器）和NOT门组成，并将输入化学物质转换为布线分子;2） 内层，它由与相应布尔公式中的子句一样多的 TU 组成。如果电路按照SOP公式设计，则内层中的每个子句都将在所谓的分布式输出架构中产生电路输出（例如荧光）。如果使用和积（POS）公式，则需要3）最后一层，它将包含一个从内层收集布线分子的乘法门。
总体而言，在合成生物学中，可以为同一电路设计许多不同的方案。它们在TU和连接分子的数量和种类上有所不同。为了选择在酵母细胞中实现的最简单解决方案，每个电路设计都与复杂性评分S相关联，定义为
<img alt="Equation 1" src="/files/ftp_upload/64539/64539eq01.jpg" style="margin: auto;">
其中 A 代表激活剂的数量， R 代表阻遏剂的数量， a 是反义RNA分子的数量。如果电路中没有激活剂或抑制因子，则它们对S的贡献为零。因此，当需要大量正交转录因子时，在实验室中实现电路方案（高S）更加困难。这意味着新的活化器和抑制器应 重新 设计，以实现数字电路内部的完整布线。原则上，可以使用锌指蛋白3 和TAL效应子4 作为模板来组装新型DNA结合蛋白。但是，此选项似乎过于艰巨和耗时;因此，人们应该主要依靠小RNA和翻译调控来完成复杂的基因回路。
最初，这种方法是为了在细菌中制造数字电路而开发的。事实上，在真核细胞中，比起反义RNA，更适合谈论microRNA（miRNA）或小干扰RNA（siRNA）5。然而，RNAi途径不存在于 酿酒酵母中。因此，应该选择完全转录网络。假设一个电路需要五个激活器和五个抑制器;其复杂性分数为 S = 32。通过将10个转录因子替换为融合到激活域（AD）的单个dCas96 （核酸酶缺陷型Cas9），可以降低电路复杂性。如7所示，当在TATA盒和TSS（转录起始位点）之间结合启动子时，dCas9-AD在酵母中充当阻遏剂，当在TATA盒上游结合时，作为激活剂起作用。因此，可以用单个dCas9-AD融合蛋白和10个sgRNA（单个向导RNA）替换10个转录因子，总复杂性得分为S = 11。合成十种sgRNA既快速又容易，而如前所述，组装10种蛋白质需要更长、更复杂的工作。
或者，可以使用两种正交的dCas蛋白（例如dCas9和dCas12a）：一种融合到AD，另一种裸露或与抑制结构域组合。复杂度分数只会增加一个单位 （S = 12）。因此，CRISPR-dCas系统是 酿酒酵母中构建非常复杂的基因数字电路的关键。
本文深入表征了酵母中基于dCas9和dCas12a的阻遏剂和激活剂的效率。结果表明，它们不需要大量的sgRNA来优化其活性，因此优先避免使用游离体质粒。此外，当使用募集VP64 AD拷贝的支架RNA（scRNA）时，基于dCas9的激活剂更有效。相比之下，dCas12a在直接融合到强VPR AD时效果很好。此外，根据激活剂的配置，合成活化启动子需要可变数量的靶位点（例如，使用 dCas12a-VPR 时为 3 个，dCas9-VP64 为 6 个，dCas9 和 scRNA 时只有一个）。作为阻遏因子，dCas12a在结合编码区而不是启动子时显得更敏锐。
然而，作为一个缺点，CRISPR-dCas9 / dCas12a不直接与化学物质相互作用。因此，它们在输入图层中可能没有用处。出于这个原因，已经研究了含有抗CRISPR蛋白（Acrs）的替代布尔门设计。Acrs作用于（d）Cas蛋白并抑制其工作8。因此，它们是调节CRISPR-（d）Cas系统活性的一种手段。本文深入分析了酿酒酵母II型Acrs与（d）Cas9以及V型Acrs和（d）Cas12a之间的相互作用。由于Acrs比Cas蛋白小得多，因此通过将人雌激素受体9-HBD（hER）的激素结合结构域融合到AcrIIA4，构建了对雌激素β-雌二醇有反应的NOT门。此外，在用半乳糖诱导时实现了少数表达dCas12a（-AD）的YES和NOT门和AcrVAs。目前，这些门仅用作概念证明。然而，它们也代表了深入重新思考算法的第一步，以执行酵母细胞中合成基因数字电路的计算自动设计。

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			<td></td>
		</tr>
		<tr>
			<td>Applied biosystems veriti 96-well thermal cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>4375786</td>
			<td></td>
		</tr>
		<tr>
			<td>AxyPrep DNA gel extraction kit</td>
			<td>Axygen</td>
			<td>AP-GX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSuite CS&#38;T research beads</td>
			<td>BD</td>
			<td>650621</td>
			<td>Fluorescent beads</td>
		</tr>
		<tr>
			<td>BD FACSVerse flow cytometer&#160;</td>
			<td>BD</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Sorvall ST 16R</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004380</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli competent cells (Strain DH5&#945;)</td>
			<td>Life Technologies</td>
			<td>18263-012</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL select Western Blotting detection reagent</td>
			<td>GE Healthcare</td>
			<td>RPN2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>Beijing JUNYI Electrophoresis Co., Ltd</td>
			<td>JY300C</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat 8-strip caps</td>
			<td>NEST</td>
			<td>406012</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene synthesis company</td>
			<td>Azenta Life Sciences</td>
			<td></td>
			<td>https://web.azenta.com/zh-cn/azenta-life-sciences</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A-11004</td>
			<td></td>
		</tr>
		<tr>
			<td>HiFiScript cDNA synthesis kit</td>
			<td>CWBIO</td>
			<td>CW2569M</td>
			<td>Kit used in step 6.2.2.1</td>
		</tr>
		<tr>
			<td>Lysate solution (Zymolyase)</td>
			<td>zymoresearch</td>
			<td>E1004-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse 80i fluorescence microscope</td>
			<td>Nikon</td>
			<td>-</td>
			<td>Fluorescence microscope</td>
		</tr>
		<tr>
			<td>Pipet tips&#8212;10 &#956;L</td>
			<td>Axygen</td>
			<td>T-300-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips&#8212;1000 &#956;L</td>
			<td>Axygen</td>
			<td>T-1000-B-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips&#8212;200 &#956;L</td>
			<td>Axygen</td>
			<td>T-200-Y-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSII403</td>
			<td>Addgene</td>
			<td>35436</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSII404</td>
			<td>Addgene</td>
			<td>35438</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSII405</td>
			<td>Addgene</td>
			<td>35440</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSII406</td>
			<td>Addgene</td>
			<td>35442</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSII424</td>
			<td>Addgene</td>
			<td>35466</td>
			<td></td>
		</tr>
		<tr>
			<td>pTPGI_dSpCas9_VP64&#160;</td>
			<td>Addgene</td>
			<td>49013</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 High-fidelity DNApolymerase</td>
			<td>New England Biolabs</td>
			<td>M0491</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction enzyme-Acc65I</td>
			<td>New England Biolabs</td>
			<td>R0599</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction enzyme-BamHI</td>
			<td>New England Biolabs</td>
			<td>R0136</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction enzyme-SacI-HF</td>
			<td>New England Biolabs</td>
			<td>R3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction enzyme-XhoI</td>
			<td>New England Biolabs</td>
			<td>R0146</td>
			<td></td>
		</tr>
		<tr>
			<td>Roche LightCycler 96&#160;</td>
			<td>Roche</td>
			<td>-</td>
			<td>Real-Time PCR Instrument</td>
		</tr>
		<tr>
			<td>S. cerevisiae CEN.PK2-1C</td>
			<td>-</td>
			<td>-</td>
			<td>The parent strain. The genotype is: MATa; his3D1; leu2-3_112; ura3-52; trp1-289; 
			MAL2-8c; SUC2</td>
		</tr>
		<tr>
			<td>Stem-Loop Kit</td>
			<td>SparkJade</td>
			<td>AG0502</td>
			<td>Kit used in step 6.2.1.3</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BIO-RAD</td>
			<td>186-1096</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202</td>
			<td></td>
		</tr>
		<tr>
			<td>T5 Exonuclease</td>
			<td>New England Biolabs</td>
			<td>M0363</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0208</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0495</td>
			<td></td>
		</tr>
		<tr>
			<td>TB Green Premix Ex Taq II (Tli RNaseH Plus)(2x) (SYBR Green I dye)</td>
			<td>Takara</td>
			<td>RR820Q</td>
			<td></td>
		</tr>
		<tr>
			<td>YeaStar RNA kit</td>
			<td>Zymo Research</td>
			<td>R1002</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-estradiol</td>
			<td>Sigma-Aldrich</td>
			<td>E8875</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene digital circuits based on crispr-cas systems and anti-crispr proteins

合成基因布尔门和数字电路具有广泛的应用，从医疗诊断到环境保护。CRISPR-Cas系统及其天然抑制剂——抗CRISPR蛋白（Acrs）的发现为设计和实施 体内 基因数字电路提供了一种新工具。在这里，我们描述了一种协议，该协议遵循“设计-构建-测试-学习”生物工程循环的思想，并利用dCas9 / dCas12a及其相应的Acrs来建立小型转录网络，其中一些行为类似于 酿酒酵母中的布尔门。这些结果指出了dCas9/dCas12a作为转录因子的性质。特别是，为了实现基因表达的最大激活，dSpCas9需要与收集VP64激活域（AD）多个拷贝的工程支架RNA相互作用。相比之下，dCas12a应在C末端与强VP64-p65-RTA （VPR） AD融合。此外，两种Cas蛋白的活性不会通过增加细胞中sgRNA / crRNA的量来增强。本文还解释了如何基于CRISPR-dCas-Acr相互作用构建布尔门。人雌激素受体的 AcrIIA4 融合激素结合结构域是对 β-雌二醇有反应的 NOT 门的核心，而由诱导性 GAL1 启动子合成的 AcrVA 允许以半乳糖作为输入来模拟 YES 和 NOT 门。在后一个电路中，AcrVA5与dLbCas12a一起表现出最佳的逻辑行为。

通过RNA聚合酶III型启动子表达sgRNA/crRNA 首先，这项工作解决了 图1A所示的转录激活电路（电路1）的工程设计。它包含三个基本组成部分：1）编码yEGFP的基因（报告基因），其之前是一系列不同的合成启动子，为dCas9 / dCas12a-AD提供靶位点;2）酵母密码子优化版本的dCas9或dCas12a融合到激活域（分别为VP64和VPR），并包含一个或两个核定位序列（NLS）。两种dCas蛋白均...

Watch this Scientific Journal Video about Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins at JoVE.com

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

CRISPR-Cas系统和抗CRISPR蛋白被整合到 酿酒酵母的布尔门方案中。新的小逻辑电路表现出良好的性能，加深了对基于dCas9/dCas12a的转录因子和抗CRISPR蛋白性质的理解。

基于CRISPR-Cas系统和抗CRISPR蛋白的基因数字电路

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

我们的协议解释了如何设计、构建和分析利用二型和五型CRISPR dCas系统以及相应的抗CRISPR蛋白的酵母基因数字电路。它是组装停留，因为它收集了零标准程序来组装和测试服务中的合成转录网络。首先，制备含有20至40纳克DNA模板，1微升正向引物，1微升反向引物，5微升DNTP混合物，0.5微升DNA聚合酶，10微升5x DNA聚合酶反应缓冲液和双蒸水的反应混合物，总体积为50微升。如手稿中所述，在热循环仪上使用触地PCR程序扩增DNA序列。通过凝胶电泳分离PCR产物，并通过DNA凝胶提取试剂盒稀释琼脂糖凝胶中的DNA序列。使用Gibson组装方法，将纯化的PCR产物插入切开式穿梭载体中，方法是让等摩尔DNA混合物在50摄氏度下进入一小时。如前所述，通过触地PCR和Gibson组装方法构建dCas12a受体载体。通过用 BamH1 和 Xhol1 消化并用 T4 DNA 连接酶连接，将酵母密码子优化的 dCas12a 蛋白插入到两个新构建的受体载体中。类似地，基于pRS2403穿梭载体构建质粒，通过触地PCR和Gibson组装法表达抗CRISPR蛋白。要进行显微镜细胞检测，请将两微升细胞溶液放在载玻片上，并用盖玻片盖住。通过打开荧光光源，显微镜和计算机在荧光显微镜下观察细胞。记下荧光光源编号后，打开电脑上的显微镜软件。将载玻片放在显微镜载物台上，选择40倍物镜，同时在绿光下观察细胞。移动课程焦点旋钮，直到出现酵母细胞的轮廓，并移动精细聚焦旋钮以聚焦细胞。要检测细胞，关闭显微镜视野后切换到计算机屏幕并单击实时。等待三到五秒钟。单击捕获以拍照并保存图片。接下来，关闭计算机、显微镜和荧光光源。测量前20分钟打开FACS机器以预热激光并将20微升细胞培养物与300微升双蒸水混合。在连接到 FACS 计算机的计算机上运行 FACS 软件并创建新实验。然后设置测量参数。接下来，根据样品的激发和发射波长选择滤光片，并将采集单元数设置为10， 000。通过测量荧光珠的强度来调节FITC滤波电压，确保两个连续实验之间珠子强度的相对差异不超过5%用双蒸水洗涤机器几秒钟以去除任何可能的多余珠子。测量样品荧光强度并单击预览，同时等待三到五秒钟以获得样品进样稳定性。最后点击获取。在实验结束时再次测量珠子。检查两颗磁珠测量值的相对差值是否超过5%后，将FACS数据导出为FCS文件。打开 R 工作室并加载脚本BDverse_analysis。R 来分析 FCS 文件。将实验名称设置为 ename，将 FCS 文件存储为dir_d的目录路径，并将结果文件创建为dir_r。接下来，设置荧光通道。设置已测量的样本数。条形图和箱形图的点图的尺寸以及 x 轴和 y 轴的最大长度。通过从相应行中删除主题标签来选择评分方法。选择与所选门控方法和点图分辨率相对应的 flowSet 对象门控，该分辨率应至少等于 256。取消注释两个过滤指令，以删除荧光为负的测量值，并删除由于手稿中描述的其他实验而导致的异常值。按源并运行脚本。包含分析结果的所有文件都是在dir_r中创建的。当合成启动子上有6个lexOp靶位点时，dSpCas9-VP64的活化效率达到最佳。而对于dCas12a-VPR而言，当将三个拷贝的lexOp插入合成启动子中时，激活效率最高。位于整合质粒上的CRISPR RNA和单向导RNA的活化率比放置在游离质粒上的活化率高1.4至2.4倍，高1.1至1.5倍。RT-qPCR结果表明，无论表达系统如何，游离体载体产生的单向导RNA CRISPR RNA水平都高于整合载体。测试了复合物dSpCas9和支架RNA募集MCP-VP64的活化效率。含有单一野生型和F6MS2发夹的支架RNA分别具有5.27倍和4.3倍的活化。然而，两种发夹的组合产生了7.54倍的活化效率。使用四种不同的启动子来驱动三种二型抗CRISPR的表达，表明它们以剂量依赖性方式起作用。强启动子将荧光水平分别降低到其值的0.21、0.11和0.13。滴定曲线显示，该回路的行为类似于打结步态，开关比接近2.3。研究了五型抗CRISPR A 1将denAS-Cas12a和dLB-Cas12a作为激活剂的荧光表达从19%降低到71%其中，当pGAL1在pTEF1和遗传CYC1t-pCYC1noTata 5型抗CRISPR-A1下产生时，Acr-VA的性能波动较大。FACS机器很脆弱，因此应小心处理。细胞溶液不应太稠密，机器应保持清洁。

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SCIENTISTS IN ACTION

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

科研

生物工程

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

生物活性蛋白或多肽在水凝胶上的应用

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

基于琼脂糖组织模拟光学幽灵的漫反射光谱研究

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

一种基于液滴的单链 dna 放大术和微球 pcr 扩增法

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

新生儿小猪体内胸腺菌的活体力学试验方法

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

基于电润的数字微流体平台，用于自动酶链接免疫吸附测定

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Reconstitution of Actin-Based Motility with Commercially Available Proteins

用市售蛋白质重建基于肌动蛋白的运动

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a ...