Name: efficient pam-less base editing for zebrafish modeling of human genetic disease with zspry-abe8e
Uploaded: 2023-02-17T13:15:00
Description: Watch this Scientific Journal Video about Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e at JoVE.com

我们感谢来自Liwen Bianji（Edanz）的Barbara Garbers博士编辑了这份手稿草稿的英文文本。这项工作得到了广东省重点领域研究与发展计划（2019B030335001）、国家重点研发计划（2019YFE0106700）、国家自然科学基金（32070819，31970782）和广东省水产经济动物病原生物学与流行病学重点实验室研究基金计划（PBEA2020YB05）的支持。

本研究严格按照华南师范大学护理与使用委员会的指导方针进行。
1. 制备合成修饰的gRNA和zSpRY-ABE8e mRNA
<ol><li>根据先前的出版物37，38设计gRNA。<ol><li>优先选择 NRN 作为 PAM 序列，因为 zSpRY-ABE8e 显示 NRN PAM 的偏好高于 NYN PAM（其中 R 为 A 或 G，Y 为 C 或 T）18。确保目标腺嘌呤核苷酸处于高度活跃的编辑?...

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该协议描述了使用碱基编辑器zSpRY-ABE8e构建斑马鱼疾病模型。与传统的HDR碱基替代途径相比，该方案可以实现更高效的碱基编辑并减少插入缺失的发生。同时，该协议涉及在斑马鱼中实施最近提出的i-Silence基因敲除策略。总之，zSpRY-ABE8e将加强斑马鱼模型在疾病研究中的应用。
脱靶效应是CRISPR/Cas9系统中的常见问题。考虑到无PAM的限制，zSpRY-ABE8e的脱靶效应可能更高，即使在先...

引起错义或无义突变的单核苷酸变异（SNV）是人类基因组中最常见的突变来源1。为了确定特定的SNV是否具有致病性，并阐明其发病机制，需要精确的动物模型2。斑马鱼是良好的人类疾病模型，表现出与人类生理和遗传同源性高、发育周期短、繁殖能力强等特点和机制研究，有利于研究致病特征和机制，以及药物筛选3。
成簇规则间隔短回文重复序列（CRISPR）/Cas9系统已广泛应用于包括斑马鱼4在内的各种物种的基因组编辑。在gRNA的指导下，CRISPR/Cas9系统可以在靶位点产生DNA双链断裂（DSB），然后通过同源定向修复（HDR）途径将靶位点与供体DNA模板重组，从而实现单碱基替换。然而，这种碱基置换方法的效率相当低，因为细胞DNA修复过程主要由非同源末端连接（NHEJ）途径进行，该途径通常伴随着插入和缺失（indel）突变5。幸运的是，基于CRISPR/Cas9的单碱基编辑技术通过使用碱基编辑器显着缓解了这个问题，可以在不诱导DSB的情况下实现更高效的单碱基编辑。已经开发了两大类碱基编辑器，腺嘌呤碱基编辑器（ABEs）和胞嘧啶碱基编辑器（CBE），以实现A·T到G·C和C·G到T·A，分别为6，7，8，9，10，11。这四种碱基取代覆盖了30%的人类致病变异12。据报道，这两类碱基编辑器，包括PmCDA1，BE系统，CBE4max，ABE7.10和ABE8e，在斑马鱼中工作，特别是BE4max和ABE8e实现了高编辑活性13，14，15，16，17，18，19。
来自不同物种的Cas9蛋白，包括金黄色葡萄球菌，化脓性链球菌和犬链球菌，已经在斑马鱼基因编辑中实施，其中化脓性链球菌Cas9（SpCas9）应用最广泛20，21，22，23。然而，SpCas9只能识别具有NGG原间隔相邻基序（PAM）的目标位点，这限制了其可编辑范围，并可能导致在感兴趣的致病位点附近找不到合适的序列24。为了扩大靶标范围，已经设计了各种SpCas9变体，以通过定向进化和结构引导设计来识别不同的PAM。然而，很少有变异在动物中有效，特别是在斑马鱼中，这限制了斑马鱼在SNV相关疾病研究中的应用25，26，27，28。最近，据报道，SpCas9的两个变体SpG和SpRY，具有不太严格的PAM限制（用于SpG的NGN和用于SpRY的NNN，对NRN的偏好高于NYN）在人类细胞和植物中表现出高编辑活性29，30，31，32。随后，SpG和SpRY以及它们介导的一些碱基编辑器，如SpRY介导的CBE和SpRY介导的ABE，也被报道在斑马鱼中起作用，这将加强斑马鱼模型在SNV相关疾病的机制研究和药物筛选中的应用18，33，34，35.此外，i-Silence被提出作为一种有效且准确的基因敲除策略，通过ABE介导的起始密码子从ATG到GTG或ACG36的转换。i-Silence策略和SpRY介导的碱基编辑器zSpRY-ABE8e的结合为疾病建模提供了一种新方法18。该协议演示了如何在斑马鱼中使用zSpRY-ABE8e进行基因编辑，以使用i-Silence策略构建tsr2（M1V）模型。对斑马鱼模型中出现的编辑效率和表型进行了评估和分析。

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			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td>1.5% Agarose used to make an injection plate</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries&#160;</td>
			<td>Harvard Apparatus</td>
			<td>BS4 30-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dishes&#160;</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>ClonExpress Ultra One Step Cloning Kit</td>
			<td>Vazyme</td>
			<td>C115</td>
			<td>Kit for Infusion clone&#160;</td>
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		<tr>
			<td>Codon optimization service</td>
			<td>Sangon Biotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drummond Microcaps</td>
			<td>Drummond Microcaps</td>
			<td>P1299-1PAK</td>
			<td>Length:32 mm, capacity:0.5 &#956;L</td>
		</tr>
		<tr>
			<td>EasyEdit gRNA service</td>
			<td>GenScript</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Scientific Instrument</td>
			<td>11254-20</td>
			<td>Used to break meedle</td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller&#160;</td>
			<td>Stutter Instrument</td>
			<td>P-97</td>
			<td>Used to pull the glass capillaries</td>
		</tr>
		<tr>
			<td>HotStart Taq PCR StarMix</td>
			<td>Genstar</td>
			<td>A033-101</td>
			<td>Used for PCR reaction</td>
		</tr>
		<tr>
			<td>Intelligent artificial climate box</td>
			<td>TENLIN</td>
			<td>PRX-1000A</td>
			<td>Used to culture zebrafish embryos</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0512</td>
			<td>Used to fix zebrafish when photographing</td>
		</tr>
		<tr>
			<td>Microloader pipette tips&#160;</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
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			<td>mMACHINE kit&#160;</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Mut Express II Fast Mutagenesis Kit V2</td>
			<td>Vazyme</td>
			<td>C214-01</td>
			<td>Kit for site-directed mutagenesis</td>
		</tr>
		<tr>
			<td>Pneumatic Microinjector</td>
			<td>ZGene Biotech</td>
			<td>ZGPCP-1500 PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td>pT3TS-zSpCas9</td>
			<td>Addgene</td>
			<td>46757</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy FFPE kit&#160;</td>
			<td>Qiagen</td>
			<td>73504</td>
			<td>Kit for RNA purification</td>
		</tr>
		<tr>
			<td>Sanger Sequencing service</td>
			<td>Sangon Biotech</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Sodium hydroxide, granular</td>
			<td>Sangon</td>
			<td>A100173-0500</td>
			<td>NaOH used for genome extraction</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus&#160;</td>
			<td>SZX10</td>
			<td>Used for photograph of phenotype</td>
		</tr>
		<tr>
			<td>SZ Series Zoom Stereo Microscope</td>
			<td>CNOPTEC</td>
			<td>SZ650</td>
			<td></td>
		</tr>
		<tr>
			<td>T3 mMESSAGE</td>
			<td>Ambion</td>
			<td>AM1348</td>
			<td>Kit for in vitro transcription&#160;</td>
		</tr>
		<tr>
			<td>TIANprep Mini Plasmid Kit</td>
			<td>TIANGEN</td>
			<td>DP103-03</td>
			<td>Kit for plasmid extraction kit</td>
		</tr>
		<tr>
			<td>TIANquick Mini Purification Kit</td>
			<td>TIANGEN</td>
			<td>DP203-02</td>
			<td>Kit for purification for linearized plasmid</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>Used to anesthetize zebrafish</td>
		</tr>
		<tr>
			<td>Tris (hydroxymethyl) aminomethane</td>
			<td>Sangon</td>
			<td>A600194-0500</td>
			<td>Component of Tris&#183;HCl used for genome extraction</td>
		</tr>
		<tr>
			<td>XbaI</td>
			<td>New England Biolabs</td>
			<td>R0145S</td>
			<td>Restriction endonuclease used for plasmid linearization</td>
		</tr>
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efficient pam-less base editing for zebrafish modeling of human genetic disease with zspry-abe8e

CRISPR/Cas9技术增加了斑马鱼在模拟人类遗传疾病、研究疾病发病机制和药物筛选方面的价值，但原间隔邻基序（PAM）的限制是创建由单核苷酸变异（SNV）引起的人类遗传疾病的准确动物模型的主要障碍。到目前为止，一些具有广泛PAM兼容性的SpCas9变体已经在斑马鱼中显示出效率。优化的SpRY介导的腺嘌呤碱基编辑器（ABE）、zSpRY-ABE8e和合成修饰的gRNA在斑马鱼中的应用实现了高效的腺嘌呤-鸟嘌呤碱基转化，不受PAM限制。这里描述的是一种使用zSpRY-ABE8e在斑马鱼中进行高效腺嘌呤碱基编辑的协议，而没有PAM限制。通过将zSpRY-ABE8e mRNA和合成修饰的gRNA的混合物注入斑马鱼胚胎中，构建了具有精确突变的斑马鱼疾病模型，该模型模拟了TSR2核糖体成熟因子（tsr2）的致病位点。该方法为建立准确的疾病模型以研究疾病机制和治疗方法提供了有价值的工具。

据报道， TSR2 的突变会导致钻石黑扇贫血（DBA）42。在这里，使用i-Silence策略构建了 具有tsr2 （M1V）突变的DBA斑马鱼模型。使用zSpRY-ABE8e将斑马鱼 tsr2 起始密码子的腺嘌呤成功转化为鸟嘌呤（图3）。
对 Sanger 测序结果的 EditR 分析表明， tsr2 起始密码子的腺嘌呤碱基存在 A/G 重叠（图 4）?...

Watch this Scientific Journal Video about Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e at JoVE.com

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

该协议描述了如何在没有PAM限制的情况下进行有效的腺嘌呤碱基编辑，以使用zSpRY-ABE8e构建精确的斑马鱼疾病模型。

使用 zSpRY-ABE8e 对斑马鱼进行人类遗传病建模的高效无 PAM 碱基编辑

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but ...

该协议允许研究人员建立许多以前不可用的斑马鱼疾病相关单核苷酸变体模型。PAM 限制是创建准确动物模型的主要障碍，zSpRY-ABE8e 能够以低换位和宽松的 PAN 限制实现腺嘌呤到鸟嘌呤碱基的高效转化。该工具可用于构建精确的斑马鱼模型，用于研究与单核苷酸海洋相关的致病特征疾病机制，以及该疾病的药物筛选。首先，设置微量移液器拉拔器系统并预热至少 30 分钟。要拉动玻璃毛细管，请选择一个程序，将斜坡测试值设置为热量，50 设置为拉力，速度为 100，时间设置为 50，压力设置为 30。接下来，安装毛细管，确保它位于凹槽中。按拉动开始/停止“运行程序。为了保护拉动的毛细管，将它们插入含有间隙的泡沫中，保持针头悬浮。对于实验，使用手稿中描述的zSpRY-ABE8e mRNA，并在两端用2'O-甲基硫代磷酸酯轻松编辑sgRNA。对于gRNA设计，优先选择NRN作为PAM序列，因为zSpRY-ABE8e显示NRN PAM的偏好高于NYN PAM。 输入目标腺嘌呤核苷酸处于沿着原间隔条从第三核苷酸到第九核苷酸的高度活跃编辑窗口。在注射前一天晚上设置繁殖池。插入一个分隔器，在每个水箱中放置两条雌性和一条雄性斑马鱼。在每个水箱上盖上盖子。第二天早上，使用不含RNA酶的吸头，试管和手套，将解冻的mRNA和EEgRNA在冰上混合至最终浓度分别为每微升400和200纳克。要配置气动微型注射器，首先，关闭气泵的分压阀。然后，打开主阀，拧下气瓶，将分压阀的压力调节到0.2兆帕。现在，打开显微注射器并将模式设置为计时器。将参数压力阀调整为每平方英寸 20 磅，将计时器值调整为 0.040 秒。使用细镊子，在体视显微镜下的角度折断拉动的玻璃毛细管的针。使用微量加载器移液器吸头，将mRNA / gRNA混合物添加到玻璃毛细管的尖端，并将针头连接到显微操纵器上。配置压力和计时器值，以使用微帽每次注射产生两纳升混合物。繁殖鱼 10 到 15 分钟后，将卵收集在过滤器中。在体视显微镜下检查细胞阶段和卵子质量。选择单细胞卵子进行注射，以提高编辑效率。将卵子排列在1.5%琼脂糖注射板上，并使用显微镜将两纳升RNA混合物注射到胚胎细胞中。用E3缓冲液将鸡蛋收集在培养皿中，并在28摄氏度下孵育。取出死卵，受精后48小时每12小时更换一次培养缓冲液。受精后48小时后，将三个随机选择的六个注射胚胎池收集到PCR管中。收集六个随机选择的对照胚胎后，使用移液器吸出残留的E3缓冲液。向胚胎中加入40微升50毫摩尔氢氧化钠。将试管放入95摄氏度的金属浴中20分钟，然后涡旋15秒。向每个试管中加入四微升一摩尔三盐酸盐，pH 8。在室温下以2000g离心管10秒钟。将上清液转移到新的PCR管中。从PCR管中取出一微升上清液并将其用于Sanger测序。然后将剩余的上清液储存在20摄氏度。要分析 Sanger 测序数据，首先将测序文件上传到上传 ab1 File“框中。将 20 个碱基对 gRNA 序列以 5'至 3' 方向输入到输入 gRNA“序列框中。在 5'start 和 3'end 框中输入相应的基本位置。通过点击预测编辑“并检查测序数据的质量来获得基础编辑效率。通过将Tsr2起始密码子的腺嘌呤转化为鸟嘌呤，构建了菱形黑扇贫血斑马鱼模型。序列数据的EditR分析表明，Tsr2起始密码子的腺嘌呤碱基存在AG重叠。与对照胚胎相比，两个日龄Tsr2胚胎的表型显示出更小的眼睛。在突变胚胎中也观察到心包肿胀。为了实现高效的基础编辑，请严格遵循前面提到的指南设计原则，这是实验成功的先决条件。准确的模型对于研究单核苷酸变异相关疾病至关重要。SprY-ABE8e的开发为精确建模提供了有效的工具，有助于促进疾病机制和治疗方法的研究。

Research

JoVE Journal

Biology

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

科研

生物学

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

In Vivo Modeling of the Morbid Human Genome using Danio rerio

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...