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Biologia

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

Published: February 17, 2023
doi:
10.3791/64977
, , , , ,
1Key Laboratory of Brain, Cognition and Education Science,Ministry of Education, 2Institute for Brain Research and Rehabilitation, Guangdong Key Laboratory of Mental Health and Cognitive Science,South China Normal University, 3Institute of Modern Aquaculture Science and Engineering, Guangdong Provincial Engineering Technology Research Center for Environmentally Friendly Aquaculture, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, School of Life Sciences,South China Normal University, 4Department of Pathology, Guangdong Provincial People’s Hospital,Guangdong Academy of Medical Sciences

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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 根据先前的出版物37，38设计gRNA。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?…

Representative Results

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）?…

Discussion

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…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

PAM-less Base EditingZSpRY-ABE8eZebrafish ModelingSingle-nucleotide VariantsDisease MechanismsDrug ScreeningMicropipette PullerMRNASgRNAPneumatic Microinjector
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Zheng, S., Liang, F., Zhang, Y., Fei, J., Qin, W., Liu, Y. Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e. J. Vis. Exp. (192), e64977, doi:10.3791/64977 (2023).
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