Name: preparing and injecting embryos of culex mosquitoes to generate null mutations using crispr/cas9
Uploaded: 2020-09-10T15:15:00.000+00:00
Duration: 7 min 45 s
Description: Watch this Scientific Journal Video about Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9 at JoVE.com

我们感谢David O'Brochta博士和昆虫遗传技术协调研究网络的所有成员，感谢他们为我们和其他人提供的关于实施遗传技术的帮助和培训。我们特别感谢香娜·阿卢维哈雷优化了微操纵方案，允许 Culex 胚胎被注射和孵化。我们还要感谢在Meuti实验室工作的本科生德文特·西蒙斯和约瑟夫·乌尔索，感谢他们帮助照顾和筛查转基因蚊子，感谢ITF的佐拉·埃尔姆卡米协助饲养和准备蚊子注射。这项工作得到了奥苏传染病研究所向MEM提供的跨学科种子赠款的支持。

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RH为昆虫转化设施工作，该设施提供昆虫基因改造服务。

该协议提出了将特定突变引入 Culex 蚊子基因组的方法，也可用于编辑其他蚊子的基因组。该协议意义重大，因为它不仅提供了如何准备注射材料的具体细节，而且还提供了如何诱导蚊子产卵以及如何准备和注射这些卵子的详细视频概述。我们还总结了如何利用雌性 Cx.pipiens 的生物学在单独的木筏上产卵，从而筛选出每个雌性子的较小比例的后代，以寻找所需的突变。这里提出的方法...

Culex蚊子分布在世界各地的温带和热带地区，传播几种致命的病毒，包括西尼罗河病毒1，圣路易斯脑炎2，以及引起犬心虫3和象虫病4的线虫。库莱克斯皮皮恩斯综合体的成员，其中包括Cx.五角星，Cx.皮皮恩斯皮皮恩斯和Cx.皮皮恩斯摩尔斯，显示其生物学的许多方面惊人的变化。例如，虽然Cx.五角星和Cx.皮皮恩斯骚扰者不能进入过冬的休眠5，6，Cx.皮皮恩斯皮皮恩斯显示强大的季节性反应，并进入糖尿病，以回应短天7，8。此外，Cx.皮皮恩斯的骚扰往往更人为的，而Cx.皮皮恩斯和Cx.五角星更动物性6。然而，在美国和世界许多其他地方，这些物种杂交，这对疾病传播有很强的影响，因为Cx.pipiens皮皮恩斯和Cx.皮皮恩斯的杂交是机会型的喂食者，将咬鸟和人类9，从而成为西尼罗河病毒的桥梁载体。研究Culex蚊子生物学的这些和其他迷人的方面受到了阻碍，部分原因是Culex蚊子在实验室中饲养比伊蚊稍微困难一些，伊蚊产生静止和耐干燥的鸡蛋10，而且功能分子工具不如Culex物种开发得那么好。
CRISPR/Cas9基因组编辑是一项强大的技术，已用于评估几个重要的蚊子物种11，12，13的生物学，包括南方房子蚊子，Culex五角星14，15，16。这项技术由詹妮弗·杜德纳和埃马努埃尔·夏彭蒂埃开发，利用细菌衍生的CRISPR相关内核糖（Cas蛋白:见范德奥斯特等人的评论）对病毒进行天然细菌防御。当注射到动物胚胎中时，Cas9蛋白质与适当的引导RNA结合，可以在基因组内产生双链断裂。这是最常通过使用Cas9蛋白质，这是复杂的指南RNA，引导内分泌酶活性到基因组的特定区域。在Cas9蛋白创造了一个特定于部位的双链断裂后，细胞机械试图使用两种机制之一来修复断裂。第一种需要通过非同源端连接（NHEJ）将两端连结在一起，这种连接容易出错，并且经常在基因组中产生框架外插入和缺失，从而产生非功能性蛋白质，从而产生淘汰突变。或者，蜂窝机械可能使用同源定向修复 （HDR）， 通过查找类似的序列来正确修复中断。类似的序列可能由生物体内的第二条染色体提供（见审查18）。但是，如果修复的序列与原始序列完全匹配，Cas9 蛋白质将能够再次切割 DNA。或者，研究人员还可以包括一个供体质粒，该质粒包含目标序列切口两侧的同源序列，该序列通常带有荧光标记蛋白、原始基因的修改版本或其他可复制并插入基因组的修饰序列，或"敲击"。
注射胚胎时，时机至关重要，在使用CRISPR/Cas9基因组编辑在昆虫中产生突变时尤其如此。这是因为Cas9蛋白质和gRNA只有在胚胎处于同步状态、细胞膜形成之前以及胚胎内可获得多个细胞核时，才具有产生突变的最大能力。对于蚊子，核在卵泡后2-4小时到达外围，取决于温度19，因此必须在此时之前成功进行微喷射。此外，Cas9蛋白质将切断任何核DNA，它可以访问，这样，从注射产生的个体将包含细胞的马赛克，有些有所需的突变，而另一些没有。为了成功遗传这些突变，Cas9蛋白质必须切割存在于生殖系中的DNA，从而产生未来的卵子和精子。为了确保在生殖系中产生突变，最好将所有材料注射到靠近胚胎中极细胞位置的位置的地方，而胚胎是昆虫生殖系的祖细胞。极细胞位于 Culex 胚胎20的后端附近。除了注射胚胎外，还必须制定一个仔细的交叉和筛选后代的计划，以便检测所需的突变。
该协议描述了如何生成gRNA，并将其与Cas9蛋白复合，以准备注射混合物，以及如何诱导 Culex皮皮恩斯 雌性蚊子产卵，以及如何准备和注射这些卵子为CRISPR/Cas9介导的基因组编辑。此外，我们描述如何后方，交叉和屏幕注射胚胎及其后代，以确认所需的突变已经获得。使用这个协议，我们产生了一个感兴趣的基因， 周期，在 Culex皮皮恩斯的巴克耶菌株的空突变。这种菌株最初于2013年从俄亥俄州哥伦布市的野外采集蚊子中建立，由Meuti实验室维持。该协议可用于其他研究，要求CRISPR/Cas9基因组编辑在 Culex 蚊子，以及其他蚊子物种，更笼统地，是相关的使用CRISPR/Cas9基因组编辑到任何昆虫物种。

<table><tbody><tr><td>Artificial Membrane Feeder</td><td>Hemotek</td><td>SP5W1-3</td><td>Company location: Blackburn, UK</td></tr><tr><td>ATP</td><td>Invitrogen</td><td>18330019</td><td>Company location: Carlsbad, CA, USA</td></tr><tr><td>Borosilicate glass mirocapillary tubes, 1 mm outer diameter</td><td>World Precision Instruments</td><td>1B100-6</td><td>Company Location: Sarasota, FL, USA</td></tr><tr><td>BV10 Needle Beveler</td><td>Sutter Instruments</td><td>BV-10-B</td><td>Company Location: Nobato, CA, USA</td></tr><tr><td>Whatman Circular filter paper (12.5 cm)</td><td>Sigma Aldrich</td><td>WHA1001125</td><td>Company Location: St. Louis, MO, USA</td></tr><tr><td>Conical tube (50 mL)</td><td>Thermo Fisher Scientific</td><td>339652</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Fisherbrand course filter paper with fast flow rate</td><td>Thermo Fisher Scientific</td><td>09-800</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Cover glass (24 x 40 mm)</td><td>Thermo Fisher Scientific</td><td>50-311-20</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Dental dam</td><td>Henry Schein Inc</td><td>1010171</td><td>Company Location: Melville, NY USA</td></tr><tr><td>Scotch double-sided tape</td><td>Thermo Fisher Scientific</td><td>NC0879005</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>FemtoJet 4i microinjector</td><td>Eppendorf</td><td>5252000021</td><td>Company Location: Hamburg, Germany</td></tr><tr><td>Glass vial (2 dram)</td><td>Thermo Fisher Scientific</td><td>033401C</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Halocarbon oil</td><td>Sigma Aldrich</td><td>H8898-50ML</td><td>Company Location: St. Louis, MO, USA</td></tr><tr><td>P-2000 Laser Needle Puller</td><td>Sutter Instruments</td><td>P-2000/G</td><td>Company Location: Nobato, CA, USA</td></tr><tr><td>Parafilm</td><td>Thermo Fisher Scientific</td><td>50-998-944</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>PC-100 Weighted Needle Puller</td><td>Narishige</td><td>PC-100</td><td>This is compatabile with the earlier PC-10 model, which has been discontinued. Company Location: Amityville, NY, USA</td></tr><tr><td>Phire Direct PCR Kit</td><td>Thermo Fisher Scientific</td><td>F140WH</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Kodak Photo-Flo (1%)</td><td>Thermo Fisher Scientific</td><td>50-268-05</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Quartz glass mirocapillary tubes, 1 mm outer diameter</td><td>Capillary Tube Supplies Limited</td><td>QGCT 1.0</td><td>Company Location: Cornwall, UK</td></tr><tr><td>Guide-it&amp;trade; sgRNA Screening Kit</td><td>Takara, Bio USA</td><td>632639</td><td>This kit allows you to determine if gRNAs cut DNA sequences in vitro. Company Location: Mountain View, CA, USA</td></tr><tr><td>Sigmacote</td><td>Sigma Aldrich</td><td>SL2-100ML</td><td>Company Location: St. Louis, MO, USA</td></tr><tr><td>Small petri dishes (35X10 mm)</td><td>Thermo Fisher Scientific</td><td>50-190-0273</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Sodium citrate chicken blood</td><td>Lampire biologicals</td><td>7201406</td><td>Company Location: Everett, PA, USA</td></tr><tr><td>Fisherbrand Square petri dish (10 cm x 10 cm)</td><td>Thermo Fisher Scientific</td><td>FB0875711A</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Tegaderm</td><td>Henry Schein Inc.</td><td>7771180</td><td>Company Location: Melville, NY USA</td></tr><tr><td>Tropical fish food</td><td>Tetramin</td><td>N/A</td><td></td></tr><tr><td>Whatman filter paper</td><td>Thermo Fisher Scientific</td><td>09-927-826</td><td>Company Location: Waltham, MA, USA</td></tr><tr><td>Whatman filter paper, 4.25 cm</td><td>Sigma Aldrich</td><td>1001-042</td><td>Company Location: St. Louis, MO, USA</td></tr></tbody></table>

preparing and injecting embryos of culex mosquitoes to generate null mutations using crispr/cas9

Culex 蚊子是多种疾病的主要传播媒介，这些疾病对人类和动物健康产生了负面影响，包括西尼罗河病毒和由犬心虫和象虫病等纤维线虫引起的疾病。最近，CRISPR/Cas9基因组编辑被用来诱导现场定向突变，通过注射一种Cas9蛋白，该蛋白质已经与导引RNA（gRNA）复合成几个昆虫物种的新鲜胚胎，包括属于 阿诺菲勒斯 家族和 伊蚊的蚊子。操纵和注射 Culex 蚊子稍微困难一些，因为这些蚊子将卵子直立在木筏上，而不是像其他种类的蚊子那样单独。在这里，我们描述如何设计gRNA，用Cas9蛋白复合它们，诱导 Culex皮皮恩斯 雌性蚊子产卵，以及如何准备和注射新铺设的胚胎与Cas9/gRNA进行微注射。我们还描述了如何饲养和筛选注射蚊子的所需突变。具有代表性的结果表明，该技术可用于诱导 Culex 蚊子基因组中的部位定向突变，并稍作修改，也可用于在其他蚊子物种中产生空突变体。

使用上述协议，我们成功地注射了Cx.pipiens的胚胎，并观察到注射胚胎的存活率很高（+55%，图1）。早期的试验存活率较低，可能是因为卵泡前部附着在医用敷料条上，防止蚊子幼虫从胆汁中逸出并成功游入水中。确保前端延伸到医疗敷料条外，大大提高了幼虫的存活率，并导致高质量的后代，能够发展到成年和繁殖（图2B）。
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Watch this Scientific Journal Video about Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9 at JoVE.com

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9越来越多地用于非模型生物体的基因功能特征。该协议描述了如何产生 Culex皮皮恩斯的淘汰线，从准备注射混合，获得和注射蚊子胚胎，以及如何后方，交叉和屏幕注射蚊子及其后代所需的突变。

准备和注射 Culex 蚊子的胚胎，使用CRISPR/Cas9产生空突变

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

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Research

JoVE Journal

Biology

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

6.8K 次观看.  The Ohio State University. CRISPR/Cas9越来越多地用于非模型生物体的基因功能特征。该协议描述了如何产生 Culex皮皮恩斯的淘汰线，从准备注射混合，获得和注射蚊子胚胎，以及如何后方，交叉和屏幕注射蚊子及其后代所需的突变。

视频: Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

科研

遗传学

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

This protocol describes microinjection procedures for Culex quinquefasciatus embryos that are optimized to work with CRISPR/Cas9 gene editing tools. This technique can efficiently generate site-specific, heritable, germline mutations that can be used for building genetic technologies in this understudied disease vector.

胚胎微注射技术，用于在库莱克斯五角星高效位点特异性突变体

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, ...

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

沙飞（木薯）胚胎微注射为CRISPR/Cas9穆塔赫内西

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.

The protocols reported here illustrate three alternative ways to assess the performance of genetically-engineered mosquitoes destined for vector control in laboratory-contained small cage trials. Each protocol is tailored to the specific modification the mosquito strain bears (gene drive or non-gene drive) and the types of parameters measured.

基因工程按蚊的小笼实验室试验

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control ...

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

数字液滴PCR检测转基因按蚊种群中的Indels突变

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA ...

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. 
High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

使用埃及伊蚊的高分辨率熔融分析进行CRISPR/Cas9诱变后的Indel检测

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases ...

Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.

Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology.

使用 CRISPR-Cas9 对埃及黄热病蚊子进行基因组编辑

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

基于CRISPR/Cas9技术构建迁徙蝗虫纯合突变体

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...