CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎
Summary
本文介绍了一种简单高效的 CRISPR/Cas9 系统在沟鲶胚胎基因编辑中的微注射协议。在这个协议中, 引导 rna 和 Cas9 蛋白被微量到一细胞胚胎的卵黄中。通过敲出两条沟鲶免疫相关基因来验证这个协议。
Abstract
整个基因组的渠道鲶鱼,鮰松毛虫, 已排序, 导致更多的机会研究渠道鲶鱼基因功能。基因敲除已被用来研究这些基因功能在体内。聚类定期 interspaced 短复发重复/CRISPR 相关蛋白 9 (CRISPR/Cas9) 系统是一个强大的工具, 用于编辑基因组 DNA 序列, 以改变其功能。传统的方法是通过显微注射将 CRISPR/Cas9 mRNA 引入单细胞胚胎, 这在鲶鱼中可能是一个缓慢而低效的过程。本文介绍了 CRISPR/Cas9 蛋白微注射鲶鱼胚胎的详细方案。简单地, 收集卵子和精子, 然后进行人工受精。受精卵被转移到含有 Holtfreter 溶液的培养皿中。注射量被校准, 然后引导 RNAs/Cas9 靶向的收费/白细胞介素1受体域包含的适配器分子 (TICAM 1) 基因和李结合凝集蛋白 (RBL) 基因微量到卵黄的一细胞胚胎。基因剔除是成功的, indels 通过 DNA 测序得到证实。由于这些突变的预测蛋白质序列的变化包括 frameshift 和截断蛋白质由于过早停止密码。
Introduction
微注射是一种常用的实验室技术, 通过玻璃毛细管1将少量的物质 (如 DNA、RNA、蛋白质和其他大分子) 输送到细胞或胚胎中。显微注射是使用特殊的设备设置, 包括微量, 微, 和显微镜2。这项技术已经被研究者用来通过转基因、基因敲除和基因治疗来改变许多生物体, 目的是了解细胞内组件的动力学3,4,5。
沟鲶 (鮰松毛虫) 是美国水产养殖和休闲捕鱼活动中最流行的鲶鱼种类。越来越需要研究沟鲶的功能基因组学, 而沟鲶完整基因组测序提高了基因组编辑工具的应用价值6,7。了解基因功能不仅可以丰富对鲶鱼进行的研究, 而且还能导致更有效的基因改良计划, 以提高鲶鱼产业。一旦确定了某一特定性状的关键基因, 它们就可以通过基因组编辑来改进鲶鱼的生产, 从而产生有益的等位基因, 选择这些等位基因, 抑制有害等位基因, 转移通过转基因的有益等位基因, 或这些选项的一些组合。将不同品种的不同商业利益性状的最佳基因结合在一起, 将大大提高鱼类生产作业的生产率和利润率8。
基因敲除是一种直接的方法来研究基因功能在体内。DNA 水平的突变将被后代继承, 这将有助于研究它们在不同世代的影响。不同的基因组编辑工具已经开发, 包括锌指核酸 (ZFNs), 转录激活剂样效应核酸 (TALENs), 和聚集定期 interspaced 短复发重复/CRISPR 相关蛋白 9 (CRISPR/Cas9) 系统9,10,11,12。
CRISPR/Cas9 系统是一个强大的, 有效的工具, 已被用来编辑基因组 dna 序列包括在鱼通过 RNA 引导的站点特定的 dna 劈裂5,13。该系统由一个引导 RNA (gRNA) 组成, 它决定了基因组中的目标序列和 DNA 切酶, Cas9。CRISPR/Cas9 系统可以被设计成针对基因组中的任何序列, 具有优于 ZFNs 和 TALENs 的几个优点: (1) 更低的成本 (2) 更容易的工程 (3) 更具体的指南 RNA 绑定到目标序列, 并减少离靶突变 (4)多个序列可以同时针对不同的 gRNA (5) 基因的高诱变率, 不能由 TALENs 突变, 和 (6) 改善系传输率的突变高达6倍, 相比 ZFNs 和 TALENs14, 15、16、17、18。
微注射的主要替代方法是电穿孔, 其中电动脉冲应用于胚胎或细胞, 以提高膜通透性和生物分子的摄取量19,20。一些转基因鱼的产生使用电穿孔, 如鳉 (Oryzias latipes), 斑马鱼 (斑马鱼), 鳞鲑 (虹 tshawytscha), 沟鲶, 海鲷 (鲷 sarba), 并鲤鱼 (鲤鱼)21,22,23,24,25,26。
电穿孔已被用来提供质粒 DNA, RNA, 和 Cas9 蛋白的基因敲除。在哺乳动物细胞中, Cas9/gRNA 质粒 DNA、Cas9 mRNA/gRNA 和 Cas9 蛋白/gRNA 配合物通过电穿孔传递, 而诱变率最高的是使用 Cas9 蛋白/gRNA 配合物来测试大多数电穿孔条件下的27。在海脊索动物 (Ciona 性) 中, TALENs 表达的构造被 electroporated 成受精卵, 以诱导多种基因的敲除28。ZFNs 表达质粒结构是 electroporated 在沟鲶中敲出黄体生成素的途径29。然而, 一旦引入细胞, 质粒结构将需要转录到 rna 和翻译成功能蛋白之前, 他们可以针对预期的 DNA 序列, 这将可能延迟的时间, 与微注射 rna/蛋白.随着胚胎的发育, 延迟诱变增加了注射嵌的形成。此外, 转基因表达不太可能通过微注射来达到表达水平, 降低诱变效率。
作为一种将基因组编辑工具引入细胞的方法, 微注射在电穿孔方面有几个优点。基因组编辑分子可以通过显微注射30可靠地引入细胞或胚胎。需要较少的注塑材料。很容易确定注入的材料的数量。更高的变异率与低嵌在创建者鱼可以达到, 将改进系传输变异到 F1。由于突变率较高, 需要分析生产第一代鱼的创始人较少。在电穿孔, 更多的创始人鱼需要分析, 这将增加成本。然而, 沟鲶微量胚的生存率低于 electroporated, 但可以通过注入更多的胚胎31,32来克服。在沟鲶中, 卵黄微量胚的存活范围从16到55% 不等, 这取决于所针对的基因和 gRNA/Cas9 蛋白的用量32。
定期注射鲶鱼胚胎在技术上要求和耗时, 然而, 一个快速和有效的 CRISPR/Cas9 蛋白显微注射协议提出的沟鲶胚胎。这一协议需要更少的时间和专业知识, 因为 CRISPR/Cas9 蛋白溶液被注射到一个细胞胚胎的蛋黄。数以百计的受精卵可以在1小时内注射 (大约是第一次发生细胞分裂所需的时间)。为了验证该协议, 在沟鲶、toll/白细胞介素1受体域内含有的适配器分子 (TICAM1) 基因和李结合凝集蛋白 (RBL) 基因中, 两种病易感性相关基因被淘汰。TICAM 1 参与了由 toll 样受体 (TLR) 3 启动的信号通路。在沟鲶, TICAM 1 是显着上调以下细菌的挑战与爱德华 ictaluri, 而它是 downregulated 在蓝鲶, 一个物种耐E. ictaluri33。RBL 在早期感染中起重要作用,杆菌柱, 杆菌病的致病剂, 在上调敏感的通道鲶鱼菌株中记录了急性和强健的杆菌, 与杆菌电阻应变34。
Protocol
Representative Results
Discussion
本文介绍了一种用于实现基因敲除的沟鲶胚显微注射的详细方案。CRISPR/Cas9 蛋白在蛋黄中的注射更简单, 节省时间, 不需要广泛的训练时, microinjecting blastodisc 的一细胞胚胎, 这是最常见的技术, 大多数基因转移和基因编辑与鱼30,42. 目前的协议证明是成功的诱导 indels 沟鲶 TICAM 1 和 RBL 基因。这些可靠和有效的程序为今后在沟鲶中产生基因敲除奠定了基础。…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究是由美国农业部-NIFA 奖2015-67015-23488 到罗杰锥。作者感谢 Dr. 的描述在视频中的育雏股票选择标准。艾哈迈德 Elaswad 和卡里姆希望感谢埃及文化和教育局在华盛顿特区的资助他们的博士研究。
Materials
| Reproboost implant | Center of Marine Biotechnology | Luteinizing hormone releasing hormone analog (LHRHa) for induction of ovulation in channel catfish females | |
| TRICAINE-S | Western Chemical. Inc. | For sedation of brood stock fish during hormone injection and egg stripping. | |
| Phenol red | Sigma-Aldrich | P0290 | 0.5%, sterile filtered |
| Stereo microscope | Olympus | 213709 | For visualizing the eggs during microinjection |
| Microinjector | ASI-Applied Scientific Instrumentation | Model MPPI-3 | For the delivery of the injection material into the embryos |
| Micromanipulator | ASI-Applied Scientific Instrumentation | Model MM33 | For holding and controlling the movement of the injection needle. |
| Eppendorf Microloader | Eppendorf | 5242956.003 | For loading injection solution into microinjection needles. |
| Vertical needle puller | David Kopf Instruments | Model 720 | For pulling microinjection needles |
| Cas9 protein | PNA Bio Inc. | CP01 | Recombinant Cas9 protein from Streptococcus pyrogenes. |
| Expand High FidelityPLUS PCR System | Roche Diagnostics, USA | For PCR and amplification of DNA templates to be used in gRNA preparation | |
| Borosilicate glass capillaries | Fisher Scientific | 1 mm outer diameter (OD), for making microinjection needles. | |
| Petri dish | VWR | 25384-302 | For holding the embryos during the microinjection. |
| Crisco | The J.M. Smucker Company | Vegetable shortening for coating spawning pans and petri dishes. | |
| Holtfreter`s solution | Home Made | 59 mM NaCl, 0.67 mM KCl, 2.4 mM NaHCO3, 0.76 mM CaCl2, 1.67 mM MgSO4 to incubate the microinjected embryos till hatch. | |
| Doxycycline hyclate USP (monohydrate) | Letco Medical | 690904 | Antibiotic added to Holtfreter's solution to 10 ppm to prevent bacterial infections. |
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