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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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생물학

沉默的火花：CRISPR/Cas9基因组编辑在弱电鱼

Published: October 27, 2019
doi:
10.3791/60253
, , , ,
1Department of Integrative Biology,Michigan State University, 2Faculty of Life Sciences, Unit of Biology and Ecology of Fishes,Humboldt University, 3Department of Biology,Cape Breton University

Summary

在这里，提出了一个协议，以生产和后CRISPR/Cas9基因组敲除电鱼。详细列出了运动和生物形态所需的分子生物学、育种和饲养要求，以及生产 Cas9 诱导 Indel F0幼虫的注射技术。

Abstract

在脊椎动物的进化史上，电接收和电发生已经发生了变化。这些独立衍生的表型具有惊人的收敛性，它们有着共同的遗传结构。这也许最好的例证是健身房和莫米里德的众多收敛特征，这两种物种丰富的电镀层能够产生和检测弱电场，被称为弱电鱼。自从发现弱电鱼用电来感知周围环境和交流以来的50年里，越来越多的科学家对发展、系统和电路神经科学的演变有了巨大的见解，细胞生理学、生态学、进化生物学和行为学。最近，电鱼的基因组资源激增。这些资源的使用已经促进了对这些物种基因型和表型之间联系的重要见解。将基因组学数据与弱电鱼表型数据集成的一个主要障碍是目前缺乏功能基因组学工具。我们在这里报告一个完整的协议，执行CRISPR/Cas9诱变，利用内源性DNA修复机制在弱电鱼。我们证明，该协议在莫米里德物种布诺米鲁斯小虫和健身房，通过使用CRISPR/Cas9来靶向第一个外显子中的印贝和点突变，同样有效。钠通道基因scn4aa。使用该协议，从两个物种获得胚胎和基因型，以确认在钠通道scn4aa的第一个外向的预测突变存在。与未注射大小匹配的对照组相比，记录显示电器官放电振幅降低，证实了敲除成功的表型。

Introduction

在脊椎动物的进化史上，电接收和电发生已经发生了变化。两个谱系的远发鱼，骨状和硅状，平行地进化电接收，和五个谱系的远程（金体素，莫米里德，和属星体，马拉普特鲁鲁斯，和西诺多特斯）并联进化电发生。这些独立衍生的表型具有惊人的收敛性，它们共享着一个共同的遗传结构1，2，3。

这也许最好的例证是，健身房和莫米里德的众多收敛特征，这两个物种丰富的电镀层，产生和检测弱电场，被称为弱电鱼。在发现弱电鱼利用电来感知周围环境和交流的50年里，越来越多的科学家对发展的演变有了巨大的了解。 ，6， 系统和电路神经科学7，8，9，10，细胞生理学11，12，生态学和能量13，14，15，16，17，行为18，19，和宏观进化3，20，21.

最近，电鱼1、22、23、24、25、26的基因组、转录组和蛋白酶资源激增。 27，28.这些资源的使用已经产生了关于基因型和表型在这些物种1，2，3，28，29之间的联系的重要见解 ， 30.将基因组学数据与弱电鱼表型数据整合的一个主要障碍是目前缺乏功能基因组学工具31。

其中一个工具是最近开发的聚类定期间隔短帕林德罗次，与Cas9内分酶（CRISPR/Cas9，CRISPR）技术配对。CRISPR/Cas9是一种基因组编辑工具，在模型32、33、34和非模型生物体35、36、37中都广泛使用。CRISPR/Cas9技术已经发展到一个点，一个实验室能够基本分子生物学可以很容易地产生基因特异性的探针称为短导RNA（sgRNA），使用非克隆方法38低成本。CRISPR 比其他敲除/敲除策略具有优势，例如变形39、40、转录激活器样效应素核酸酶（TALEN）和锌指核酸酶（ZFN），这些策略成本高昂，为每个目标基因生成非常耗时。

CRISPR/Cas9 系统通过针对基因组的特定区域（由 sgRNA 序列引导）创建基因敲除，并造成双链断裂。细胞检测到双链断裂，并优先使用非同源端连接（NHEJ）路径触发内源性DNA修复机制。此通路极易出错：在修复过程中，DNA 分子通常会在双链断裂部位插入或删除（indels）。这些印子可能导致功能丧失，原因包括 （1） 打开阅读框架中的偏移，（2） 插入过早停止的芯子，或 （3） 基因产物的关键初级结构的偏移。在此协议中，我们使用CRISPR/Cas9编辑，在弱电鱼种中使用NHEJ来靶向目标基因的点突变。虽然比其他技术更简单、更有效，但这种诱变方法预计在F0中会产生一系列表型分裂，这归因于遗传马赛克41、42、43 ， 44.

有机物的选择
为了便于今后研究弱电鱼的比较基因组学，需要选择具有代表性的品种，用于植物体和摩尔米里德的礼仪开发。在乌拉圭蒙得维的亚举行的2016年电鱼会议上，经过讨论，社区达成共识，利用已在实验室中培育且有基因组资源的物种。健身房形式布拉奇希波姆高德里奥和莫米里德布里诺米鲁斯小球被选为符合这些标准的物种。在这两个物种中，诱导和维持繁殖条件的自然线索在圈养中很容易模仿。B. gauderio，一种来自南美洲的健身房品种，具有低饲养要求的优势：鱼可以在相对较小的（4L）水箱中保持相对较高的密度。在俘虏条件下，高德里奥也有快速的代际周转。在实验室条件下，B.高德里奥可以在4个月内从卵子发育到成人。

B. 小鱼，一种来自中西部非洲的莫米里德鱼，在圈养中繁殖容易。B. 近距离比乌斯通过水族馆贸易很容易获得，已被广泛用于许多研究，现在有一些基因组资源可用。其生命周期为 1⁄3 年，具体取决于实验室条件。对于该物种，养殖需求较为密集，由于在繁殖过程中受到攻击，需要中等大小的水箱（50–100 L）。

研究其他种类的电鱼的实验室应该能够轻松地适应这一协议，只要该物种可以繁殖，并且单细胞胚胎可以收集和饲养到成年。住房、幼虫饲养和体外受精（IVF）率可能会随着其他物种的变化而变化;然而，该协议可以作为其他弱电鱼繁殖尝试的起点。

概念验证的理想基因靶点：scn4aa
弱电虫和健美鱼通过释放一种称为电器官的专用器官产生电场（电发生）。电器官放电（EODs）是电细胞中称为电细胞同时产生作用电位的结果。EOD被皮肤中的电受体阵列检测到，以产生鱼类周围环境的高分辨率电图像45。弱电鱼还能够检测其共分物的EOD波形18及其放电速率46的特征，使EOD能够另外作为类似于鸟鸣或青蛙的社会通信信号发挥作用发声47.

在莫米里德和健力酸弱电鱼的电细胞中，作用电势生成的主要成分是电压门钠通道NaV1.42。非电电电表达两个副基因副本，scn4aa和scn4ab，编码为电压门钠通道NaV1.430。在健身房和莫米里德弱电鱼系中，scn4aaa发展迅速，经历了许多氨基酸替代，影响其动力学特性48。最重要的是，scn4aaa在电器官2、3的两个谱系中都变得分门别类。scn4aa对电器官的表达相对受限，在EOD的产生中起着关键作用，使其成为CRISPR/Cas9敲除实验的理想目标，因为它具有最小的有害性多效效应。由于弱电鱼在受精后6-8天开始排出幼虫电器官（DPF），因此，scn4aa的靶向非常适合在胚胎显注后快速表示。

Protocol

此处描述的所有方法均已获得密歇根州立大学机构动物护理和使用委员会 （IACUC） 的批准。 1. 选择sgRNA靶点 注：在步骤 1.1 中为 sgRNA 的手动设计提供了一个协议。这被用于scn4aa目标选择。提供了一个附加协议，以方便此过程（步骤 1.2）使用 EFISHGENOMICS Web 门户。建议用户选择协议 1.2，它具有几个自动”检查”功能，以确保成功为自定义目?…

Representative Results

sgRNA靶点位于B.gauderio和B.b.brachyisus的scn4aa的外量1内，如第1节所述。sgRNA 的生成情况如下，如第 2 节所述。在成功选择和合成sgRNA（图1）后，对体外裂解进行了测试（图2）。然后选择在体外切割的sgRNA进行单细胞显微注射。 成年鱼被以繁殖为条件（第4.1节），然后注射产卵剂（第4.2节），然后挤压（B.gauderio?…

Discussion

弱电鱼的表型丰富性，加上最近基因组学资源的扩散，激发了弱电鱼模型中对功能基因组工具的强烈需求。该系统特别有吸引力，因为鱼类平行谱系中许多型特征的收敛进化，这些特征很容易保存在实验室中。

此处描述的协议演示了 CRISPR/Cas9 技术在同时进化电成和电接收的弱电鱼谱系中的有效性，因此代表了该模型承诺解决的一个重要步骤表型进化的比较基因组学的未来工?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢莫妮卡·卢卡斯、凯瑟琳·肖、瑞安·泰勒、贾里德·汤普森、妮可·罗比乔和霍普·希利在鱼类养殖、数据收集和早期协议开发方面所付出的英勇努力。我们还要感谢三位审评员对手稿的建议。我们相信，在发表他们的意见后，最终产品的质量会更好。这项工作由美国国家科学基金会#1644965和#1455405资助，并资助了自然科学和工程研究理事会向VLS提供DG赠款。

Materials

20 mg/mL RNA grade Glycogen Thermo Scientific R0551
50 bp DNA ladder NEB N3236L
borosilicate glass capillary with filament Sutter Instrument BF100-58-10 (O.D. 1.0mm, I.D. 0.58 mm, 10 cm length)
Cas9 protein with NLS; 1 mg/mL PNA Biology CP01
Dneasy Blood & Tissue Kit Qiagen 69506
Eppendorf FemptoJet 4i Microinjector Fisher Scientific E5252000021
Eppendorf Microloader Pipette Tips Fisher Scientific 10289651
Hamilton syringe Fisher Scientific 14-824-654 referred to as "precision glass syringe" in the protocol
Kimwipe Fisher Scientific 06-666 referred to as "delicate task wipe" in the protocol
MEGAscript T7 Transcription Kit Invitrogen AM1334
NEBuffer 3 NEB B7003S used for in vitro cleavage assay
OneTaq DNA kit NEB M0480L
Ovaprim Syndel USA https://www.syndel.com/ovaprim-ovammmlu010.html referred to as "spawning agent" in the protocol
Parafilm Fisher Scientific S37440 referred to as "thermoplastic" in the protocol
Pipette puller WPI SU-P97 sutter brand
QIAquick PCR Purification Kit Qiagen 28106
Reusable needle- requires customization Fisher Scientific 7803-02 Customize to 0.7 inches long; point style 4 and angle 25
T4 DNA polymerase NEB M0203L Use with the 10X NEB buffer that is included
Teflon coated tools bonefolder.com T-SPATULA4PIECE referred to as "polytetrafluoroethene" in the protocol
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References

  1. Gallant, J. R., et al. Genomic basis for the convergent evolution of electric organs. Science. 344 (6191), 1522-1525 (2014).
  2. Zakon, H. H., Lu, Y., Zwickl, D. J., Hillis, D. M. Sodium channel genes and the evolution of diversity in communication signals of electric fishes: convergent molecular evolution. Proceedings of the National Academy of Sciences of the United States of America. 103 (10), 3675-3680 (2006).
  3. Arnegard, M. E., Zwickl, D. J., Lu, Y., Zakon, H. H. Old gene duplication facilitates origin and diversification of an innovative communication system–twice. Proceedings of the National Academy of Sciences of the United States of America. 107, 22172-22177 (2010).
  4. Lissmann, H. W. Continuous electrical signals from the tail of a fish. Gymnarchus niloticus Cuv. Nature. 167 (4240), 201-202 (1951).
  5. Cuellar, H., Kim, J. A., Unguez, G. A. Evidence of post-transcriptional regulation in the maintenance of a partial muscle phenotype by electrogenic cells of S. macrurus. FASEB Journal. 20 (14), 2540 (2006).
  6. Modrell, M. S., Baker, C. V. Evolution of electrosensory ampullary organs: conservation of Eya4 expression during lateral line development in jawed vertebrates. Evolution & Development. 14 (3), 277-285 (2012).
  7. Hopkins, C. D. Design features for electric communication. Journal of Experimental Biology. 202, 1217-1228 (1999).
  8. Kawasaki, M. Sensory hyperacuity in the jamming avoidance response of weakly electric fish. Current Opinion in Neurobiology. 7 (4), 473-479 (1997).
  9. Bell, C. C., Han, V. Z., Sugawara, Y., Grant, K. Synaptic plasticity in a cerebellum-like structure depends on temporal order. Nature. 387 (6630), 278-281 (1997).
  10. Heiligenberg, W. . Neural Nets in Electric Fish. , (1991).
  11. Ban, Y., Smith, B. E., Markham, M. R. A highly polarized excitable cell separates sodium channels from sodium-activated potassium channels by more than a millimeter. Journal of Neurophysiology. 114 (1), 520-530 (2015).
  12. Markham, M. R., Kaczmarek, L. K., Zakon, H. H. A sodium-activated potassium channel supports high-frequency firing and reduces energetic costs during rapid modulations of action potential amplitude. Journal of Neurophysiology. 109 (7), 1713-1723 (2013).
  13. Gavassa, S., Stoddard, P. K. Food restriction promotes signaling effort in response to social challenge in a short-lived electric fish. Hormones and Behavior. 62 (4), 381-388 (2012).
  14. Sinnett, P. M., Markham, M. R. Food deprivation reduces and leptin increases the amplitude of an active sensory and communication signal in a weakly electric fish. Hormones and Behavior. 71, 31-40 (2015).
  15. Salazar, V. L., Stoddard, P. K. Sex differences in energetic costs explain sexual dimorphism in the circadian rhythm modulation of the electrocommunication signal of the gymnotiform fish Brachyhypopomus pinnicaudatus. Journal of Experimental Biology. 211, 1012-1020 (2008).
  16. Lewis, J. E., Gilmour, K. M., Moorhead, M. J., Perry, S. F., Markham, M. R. Action potential energetics at the organismal level reveal a trade-off in efficiency at high firing rates. Journal of Neuroscience. 34 (1), 197-201 (2014).
  17. Salazar, V. L., Krahe, R., Lewis, J. E. The energetics of electric organ discharge generation in gymnotiform weakly electric fish. Journal of Experimental Biology. 216 (13), 2459-2468 (2013).
  18. Hopkins, C. D., Bass, A. Temporal coding of species recognition signals in an electric fish. Science. 212 (4490), 85-87 (1981).
  19. Arnegard, M. E., Jackson, B. S., Hopkins, C. D. Time-domain signal divergence and discrimination without receptor modification in sympatric morphs of electric fishes. The Journal of Experimental Biology. 209, 2182-2198 (2006).
  20. Sullivan, J. P., Lavoue, S., Arnegard, M. E., Hopkins, C. D. AFLPs resolve phylogeny and reveal mitochondrial introgression within a species flock of African electric fish (Mormyroidea: Teleostei). Evolution. 58 (4), 825-841 (2004).
  21. Crampton, W. G. R. Effects of anoxia on the distribution, respiratory strategies and electric signal diversity of gymnotiform fishes. Journal of Fish Biology. 53, 307-330 (1998).
  22. Pinch, M., Guth, R., Samanta, M. P., Chaidez, A., Unguez, G. A. The myogenic electric organ of Sternopygus macrurus: a non-contractile tissue with a skeletal muscle transcriptome. PeerJ. 4, 1828 (2016).
  23. Lamanna, F., Kirschbaum, F., Waurick, I., Dieterich, C., Tiedemann, R. Cross-tissue and cross-species analysis of gene expression in skeletal muscle and electric organ of African weakly-electric fish (Teleostei; Mormyridae). BMC Genomics. 16, 668 (2015).
  24. Traeger, L. L., et al. Unique patterns of transcript and miRNA expression in the South American strong voltage electric eel (Electrophorus electricus). BMC Genomics. 16, 243 (2015).
  25. Salisbury, J. P., et al. The central nervous system transcriptome of the weakly electric brown ghost knifefish (Apteronotus leptorhynchus): de novo assembly, annotation, and proteomics validation. BMC Genomics. 16, 166 (2015).
  26. Lamanna, F., Kirschbaum, F., Tiedemann, R. De novo assembly and characterization of the skeletal muscle and electric organ transcriptomes of the African weakly electric fish Campylomormyrus compressirostris (Mormyridae, Teleostei). Molecular Ecology Resources. 14 (6), 1222-1230 (2014).
  27. Mate, S. E., Brown, K. J., Hoffman, E. P. Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction. Skeletal Muscle. 1 (1), 20 (2011).
  28. Swapna, I., et al. Electrostatic Tuning of a Potassium Channel in Electric Fish. bioRxiv. , (2017).
  29. Futuyma, . Evolution. Third Edition. , (2013).
  30. Thompson, A., Vo, D., Comfort, C., Zakon, H. H. Expression Evolution Facilitated the Convergent Neofunctionalization of a Sodium Channel Gene. Molecular Biology and Evolution. 31 (8), 1941-1955 (2014).
  31. Pitchers, W. R., Constantinou, S. J., Losilla, M., Gallant, J. R. Electric fish genomics: Progress, prospects, and new tools for neuroethology. Journal of Physiology Paris. , (2016).
  32. Liang, X., et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. Journal of Biotechnology. 208, 44-53 (2015).
  33. Jung, C. J., et al. Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein. Transgenic Research. 26 (2), 263-277 (2017).
  34. Liu, K., Petree, C., Requena, T., Varshney, P., Varshney, G. K. Expanding the CRISPR Toolbox in Zebrafish for Studying Development and Disease. Frontiers in Cell and Developmental Biology. 7 (13), (2019).
  35. Zu, Y., et al. Biallelic editing of a lamprey genome using the CRISPR/Cas9 system. Scientific Reports. 6, 23496 (2016).
  36. Crispo, M., et al. Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PLoS One. 10 (8), 0136690 (2015).
  37. Sun, D., Guo, Z., Liu, Y., Zhang, Y. Progress and Prospects of CRISPR/Cas Systems in Insects and Other Arthropods. Frontiers in Physiology. 8, 608 (2017).
  38. Gagnon, J. A., et al. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One. 9 (5), 98186 (2014).
  39. Kok, F. O., et al. Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish. Developmental Cell. 32 (1), 97-108 (2015).
  40. Morcos, P. A., Vincent, A. C., Moulton, J. D. Gene Editing Versus Morphants. Zebrafish. 12 (5), 319 (2015).
  41. Mehravar, M., Shirazi, A., Nazari, M., Banan, M. Mosaicism in CRISPR/Cas9-mediated genome editing. 발생학. 445 (2), 156-162 (2019).
  42. Yen, S. T., et al. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. 발생학. 393 (1), 3-9 (2014).
  43. Singh, P., Schimenti, J. C., Bolcun-Filas, E. A Mouse Geneticist’s Practical Guide to CRISPR Applications. 유전학. 199 (1), 1-15 (2015).
  44. Mianné, J., et al. Analyzing the outcome of CRISPR-aided genome editing in embryos: screening, genotyping and quality control. Methods. 121-122, 68-76 (2017).
  45. van der Emde, G., Breed, M. D., Moore, J. . Encyclopedia of Animal Behavior. 1, 16-23 (2010).
  46. Carlson, B. A., Binder, M. D., Hirokawa, N., Windhorst, U., Hirsch, M. C. . Encyclopedia of Neuroscience. , 4039-4044 (2009).
  47. Hopkins, C. D. Neruoethology of Electric Communication. Annual Reviews of Neuroscience. 11, 497-535 (1988).
  48. Arnegard, M., Zwickl, D., Lu, Y., Zakon, H. H. Old gene duplication facilitates origin and diversification of an innovative communication system- twice. Proceedings of the National Academy of Sciences of the United States of America. 107 (51), 22172-22177 (2010).
  49. Doench, J. G., et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nature Biotechnology. 32 (12), 1262-1267 (2014).
  50. Concordet, J. P., Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Resarch. 46, 242-245 (2018).
  51. Haeussler, M., et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biology. 17 (1), 148 (2016).
  52. Kirschbaum, F. Environmental factors control the periodical reproduction of tropical electric fish. Experientia. 31 (10), 1159-1160 (1975).
  53. Iwama, G. K., McGeer, J. C., Pawluk, M. P. The effects of five fish anaesthetics on acid-base balance, hematocrit, cortisol and adrenaline in rainbow trout. Canadian Journal of Zoology. 67, 2065-2073 (1989).
  54. Westerfield, M. . The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed. , (2000).
  55. Barrangou, R., Doudna, J. A. Applications of CRISPR technologies in research and beyond. Nature Biotechnology. 34 (9), 933-941 (2016).
  56. Adli, M. The CRISPR tool kit for genome editing and beyond. Nature Communications. 9 (1), 1911 (2018).
  57. Maruyama, T., et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nature Biotechnology. 33 (5), 538-542 (2015).
  58. Liu, M., et al. Methodologies for Improving HDR Efficiency. Frontiers in Genetics. 9, 691 (2018).
  59. Kirschbaum, F., et al. Intragenus (Campylomormyrus) and intergenus hybrids in mormyrid fish: Physiological and histological investigations of the electric organ ontogeny. Journal of Physiology Paris. 110, 281-301 (2016).
  60. Jao, L. E., Wente, S. R., Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proceedings of the National Academy of Science of the United States of America. 110 (34), 13904-13909 (2013).

Tags

CRISPR/Cas9Gene EditingWeakly Electric FishTranscriptomicsGenomicsMicroinjectionZebrafishSpawning AgentSperm CollectionB. GauderioB. Brachyistius

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Constantinou, S. J., Nguyen, L., Kirschbaum, F., Salazar, V. L., Gallant, J. R. Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish. J. Vis. Exp. (152), e60253, doi:10.3791/60253 (2019).
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