私たちは、デビッド・オブロフタ博士と昆虫遺伝技術調整研究ネットワークのすべてのメンバーに、彼らが遺伝技術の実施に関して私たちや他の人々に提供する助けと訓練に感謝します。我々は特に 、Culex 胚を注入し、孵化できるようにマイクロマニピュレーションプロトコルを最適化してくれたチャンナ・アルヴィヘアに感謝する。また、ムーティラボで働く学部生のデバンテ・シモンズとジョセフ・ウルソは、トランスジェニック蚊の世話とスクリーニングを支援し、ITFのゾラ・エルムカに対して、蚊の注射の飼育と準備を支援してくれたことに感謝します。この研究は、MEMに提供されたOSUの感染症研究所からの学際的種子助成金によって支えられた。

<ol>
	<li>Hamer, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culex+pipiens+(Diptera:+Culicidae):+a+bridge+vector+of+West+Nile+virus+to+humans.">Culex pipiens (Diptera: Culicidae): a bridge vector of West Nile virus to humans.</a> Journal of Medical Entomology. 45 (1), 125-128 (2008).</li><li>Bailey, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+St.+Louis+encephalitis+virus+from+overwintering+Culex+pipiens+mosquitoes.">Isolation of St. Louis encephalitis virus from overwintering Culex pipiens mosquitoes.</a> Science. 199 (4335), 1346-1349 (1978).</li><li>Sabry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+realistic+index+of+experimental+transmission+efficiency+for+Bancroftian+filariasis.">A new realistic index of experimental transmission efficiency for Bancroftian filariasis.</a> The Journal of Tropical Medicine and Hygiene. 94 (4), 283-290 (1991).</li><li>Cancrini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aedes+albopictus+and+Culex+pipiens+implicated+as+natural+vectors+of+Dirofilaria+repens+in+central+Italy.">Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy.</a> Journal of Medical Entomology. 44 (6), 1064-1066 (2007).</li><li>Wilton, D. P., Smith, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+diapause+in+three+geographic+strains+of+Culex+pipiens+(Diptera:+Culicidae).">Ovarian diapause in three geographic strains of Culex pipiens (Diptera: Culicidae).</a> Journal of Medical Entomology. 22 (5), 524-528 (1985).</li><li>Mattingly, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Culex+pipiens+complex.">The Culex pipiens complex.</a> Transactions of the Royal Entomological Society of London. 102 (7), 331-342 (1951).</li><li>Eldridge, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+temperature+and+photoperiod+on+blood-feeding+and+ovarian+development+in+mosquitoes+of+the+Culex+pipiens+complex.">The effect of temperature and photoperiod on blood-feeding and ovarian development in mosquitoes of the Culex pipiens complex.</a> The American Journal of Tropical Medicine and Hygiene. 17 (1), 133-140 (1968).</li><li>Spielman, A., Wong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+control+of+ovarian+diapause+in+Culex+pipiens.">Environmental control of ovarian diapause in Culex pipiens.</a> Annals of the Entomological Society of America. 66 (4), 905-907 (1973).</li><li>Fritz, M. L., Walker, E. D., Miller, J. R., Severson, D. W., Dworkin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+host+preferences+of+above-and+below-ground+Culex+pipiens+mosquitoes+and+their+hybrid+offspring.">Divergent host preferences of above-and below-ground Culex pipiens mosquitoes and their hybrid offspring.</a> Medical and Veterinary Entomology. 29 (2), 115-123 (2015).</li><li>Meola, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+temperature+and+humidity+on+embryonic+longevity+in+Aedes+aegypti.">The influence of temperature and humidity on embryonic longevity in Aedes aegypti.</a> Annals of the Entomological Society of America. 57 (4), 468-472 (1964).</li><li>Dong, S., Lin, J., Held, N. L., Clem, R. J., Passarelli, A. L., Franz, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+CRISPR/Cas9-mediated+genome+editing+in+the+yellow+fever+mosquito,+Aedes+aegypti.">Heritable CRISPR/Cas9-mediated genome editing in the yellow fever mosquito, Aedes aegypti.</a> PloS one. 10 (3), (2015).</li><li>Kyrou, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CRISPR-Cas9+gene+drive+targeting+doublesex+causes+complete+population+suppression+in+caged+Anopheles+gambiae+mosquitoes.">A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.</a> Nature Biotechnology. 36 (11), 1062-1066 (2018).</li><li>Liu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+an+efficient+genomic+editing+system+with+CRISPR/Cas9+in+the+vector+mosquito+Aedes+albopictus.">Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus.</a> Insect Science. 26 (6), 1045-1054 (2019).</li><li>Itokawa, K., Komagata, O., Kasai, S., Ogawa, K., Tomita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+causality+between+CYP9M10+and+pyrethroid+resistance+using+the+TALEN+and+CRISPR/Cas9+technologies.">Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies.</a> Scientific Reports. 6, 24652 (2016).</li><li>Li, M., Li, T., Liu, N., Raban, R. R., Wang, X., Akbari, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+generation+of+heritable+germline+mutations+in+the+disease+vector+Culex+quinquefasciatus+using+clustered+regularly+interspaced+short+palindrome+repeats-associated+protein+9.">Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats-associated protein 9.</a> Insect Molecular Biology. 29, 214-220 (2019).</li><li>Anderson, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+gene+editing+in+the+West+Nile+Virus+vector,+Culex+quinquefasciatus+Say.">CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say.</a> PloS one. 14 (11), (2019).</li><li>Van der Oost, J., Jore, M. M., Westra, E. R., Lundgren, M., Brouns, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-based+adaptive+and+heritable+immunity+in+prokaryotes.">CRISPR-based adaptive and heritable immunity in prokaryotes.</a> Trends in Biochemical Sciences. 34 (8), 401-407 (2009).</li><li>Heyer, W. D., Ehmsen, K. T., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+homologous+recombination+in+eukaryotes.">Regulation of homologous recombination in eukaryotes.</a> Annual Review of Genetics. 44, 113-139 (2010).</li><li>Monnerat, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anopheles+albitarsis+embryogenesis:+morphological+identification+of+major+events.">Anopheles albitarsis embryogenesis: morphological identification of major events.</a> Memorias do Instituto Oswaldo Cruz. 97 (4), 589-596 (2002).</li><li>Davis, C. W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+larval+embryogenesis+in+the+mosquito+Culex+fatigans+Wiedemann+(Diptera:+Culicidae)+and+the+sheep-fly+Lucilia+sericata+Meigen+(Diptera:+Calliphoridae).">A comparative study of larval embryogenesis in the mosquito Culex fatigans Wiedemann (Diptera: Culicidae) and the sheep-fly Lucilia sericata Meigen (Diptera: Calliphoridae).</a> Australian Journal of Zoology. 15 (3), 547-579 (1967).</li><li>Port, F., Chen, H. M., Lee, T., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+CRISPR/Cas+tools+for+efficient+germline+and+somatic+genome+engineering+in+Drosophila.">Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.</a> Proceedings of the National Academy of Sciences. 111 (29), 2967-2976 (2014).</li><li>Kistler, K. E., Vosshall, L. B., Matthews, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+with+CRISPR-Cas9+in+the+mosquito+Aedes+aegypti.">Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti.</a> Cell Reports. 11 (1), 51-60 (2015).</li><li>Gokcezade, J., Sienski, G., Duchek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+CRISPR/Cas9+plasmids+for+rapid+and+versatile+genome+editing+in+Drosophila.">Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila.</a> G3: Genes, Genomes, Genetics. 4 (11), 2279-2282 (2014).</li><li>Hwang, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+genome+editing+using+RNA-guided+nucleases.">Efficient in vivo genome editing using RNA-guided nucleases.</a> Nature Biotechnology. 31 (3), 227-229 (2013).</li><li>Basu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+end-joining+repair+for+efficient+site-specific+gene+insertion+after+TALEN/CRISPR+mutagenesis+in+Aedes+aegypti.">Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.</a> Proceedings of the National Academy of Sciences. 112 (13), 4038-4043 (2015).</li><li>Hammond, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CRISPR-Cas9+gene+drive+system+targeting+female+reproduction+in+the+malaria+mosquito+vector+Anopheles+gambiae.">A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.</a> Nature Biotechnology. 34 (1), 78-83 (2016).</li><li>Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+homology-directed+human+genome+engineering+by+controlled+timing+of+CRISPR/Cas9+delivery.">Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.</a> eLife. 3, 04766 (2014).</li></ol>

RHは昆虫の遺伝子組み換えでサービスを提供する昆虫変換施設で働いています。

このプロトコルは 、Culex 蚊のゲノムに特定の突然変異を導入する方法を提示し、他の蚊のゲノムを編集するためにも使用することができます。このプロトコルは、注射材料を準備する方法だけでなく、蚊に卵を産む方法の詳細なビデオ概要、ならびにそれらの卵を準備して注入する方法の詳細なビデオ概要を提供するという点で重要です。また、メスの Cx.ピピエン の生物学を?...

Culex蚊は、世界の温帯および熱帯地域全体に分布し、西ナイルウイルス1、セントルイス脳炎2だけでなく、イヌ心虫3および象の毛虫症を引き起こすフィラリア線虫を含むいくつかの致命的なウイルスを伝播する。Cx.クインケファシアトゥス、Cx.ピピエンスピピエンス、Cx.ピピエンス痴漢を含むCulex pipiens複合体のメンバーは、彼らの生物学の多くの側面で顕著なバリエーションを示しています。例えば、Cx.quinquefasciatusおよびCx.ピピエンス痴漢は越冬休眠5、6、Cx.ピピエンスピピエンは堅牢な季節応答を表示し、短い日7、8に応答してジアポーズに入ることができない。さらに、Cx.ピピエンス痴漢はより人類愛的である傾向があり、Cx.ピピエンとCx.キンケファシアトゥスはより動物性の6です。しかし、米国および世界の他の多くの場所で、これらの種は、Cx.ピピエンスピピエンとCx.ピピエンス痴漢のハイブリッドとして病気の伝染に強い影響を与える交雑種は日和見フィーダーであり、鳥と人間の両方を噛み、それによって西ナイルウイルスの橋梁ベクトルとして機能します。Culex蚊の生物学のこれらの側面やその他の魅力的な側面を研究することは、Culex蚊が静止と乾燥耐性卵10を生成するAedes蚊よりも実験室で飼育するのがわずかに難しく、機能的な分子ツールがCulex種のために十分に開発されていないため、部分的に妨げられております。
CRISPR/Cas9ゲノム編集は、南部の家の蚊、カレックスキンケファシアトゥス14、15、16を含むいくつかの重要な蚊種11、12、13の生物学を評価するために使用されている強力な技術です。ジェニファー・ダウドナとエマニュエル・シャルパンティエによって開発されたこの技術は、細菌由来のCRISPR関連エンドヌクレアーゼ(Casタンパク質、ファン・デル・ウーストらによるレビューを参照)によってウイルスに対する自然な細菌防御を利用しています。動物の胚に注入されると、Cas9タンパク質と適切なガイドRNAを組み合わせて、ゲノム内で二本鎖破断を生じさせることができる。これは、エンドヌクレアーゼ活性をゲノムの特定の領域に導くガイドRNAと複合体化されたCas9タンパク質を使用することによって最も頻繁に行われます。Cas9タンパク質が部位特異的な二本鎖破断を作成した後、細胞機械は2つのメカニズムのいずれかを使用して破断を修復しようとします。1つ目は、非相同の端結合(NHEJ)を通じて2つの端を結び付ける必要があり、これはエラーが起こりやすいため、ゲノムにアウトフレーム挿入および欠失を生じさせ、非機能的なタンパク質を引き起こし、ノックアウト突然変異を生じさせる。あるいは、細胞機械は、正しく破断を修復するために類似した配列を見つけることによって相同性指向の修復(HDR)を使用するかもしれません。同様の配列は、生物内の第2染色体によって提供され得る(レビュー18を参照)。しかし、修復された配列が元の配列と正確に一致する場合、Cas9タンパク質は再びDNAを切断することができます。あるいは、標的配列の切断部位の両側に相同配列を含むドナープラスミドを、代替修復配列(蛍光マーカータンパク質、元遺伝子の改変版、またはその他の改変)を含み、ゲノムにコピーして挿入したり、「ノックイン」したりすることもできます。
胚を注入する際にはタイミングが重要であり、CRISPR/Cas9ゲノム編集を使用して昆虫の突然変異を作り出す場合は特にそうです。これは、Cas9タンパク質とgRNAは、胚が間合状態にある場合、細胞膜が形成される前、および胚内で複数の核がアクセス可能である場合にのみ突然変異を生成する最大の能力を有するためである。蚊の場合、原子核は、温度19に応じて、卵子から〜2〜4時間後に周囲に到達するため、この時間までにマイクロインジェクションが成功する必要があります。さらに、Cas9タンパク質は、注射に起因する個体が細胞のモザイクを含み、所望の突然変異を有する個体が含まれるなど、アクセス可能な核DNAを切断する。これらの突然変異を正常に継承するためには、Cas9タンパク質は、将来の卵子と精子を生み出す生殖細胞系列に存在するDNAを切断する必要があります。生殖細胞系列に突然変異が発生することを確実にするためには、昆虫生殖細胞系の前駆体である胚内の極細胞の位置に近い全ての物質を注入するのが最善である。極細胞は 、Culex 胚20の後端付近に位置する。胚を注入することに加えて、所望の突然変異を検出するためには、子孫の交育とスクリーニングのための慎重な計画を策定することが不可欠である。
このプロトコルは、gRNAを生成し、注射ミックスを準備するためにCas9タンパク質でそれらを複雑にする方法だけでなく、卵を産むためにCulexピピエンのメスの蚊を誘導する方法とCRISPR / Cas9媒介ゲノム編集のためにそれらの卵を準備し、注入する方法を説明します。さらに、注射された胚の後部、横断、スクリーニングの方法とその子孫を説明し、所望の突然変異が得られたことを確認する。このプロトコルを使用して、私たちは、カレックスピピエンのバックアイ株で、関心のある遺伝子、サイクル、ヌル突然変異を生成しました。この株はもともとオハイオ州コロンバスのフィールド収集蚊から2013年に設立され、Meutiラボによって維持されています。このプロトコルは、Culex蚊および他の蚊種におけるCRISPR/Cas9ゲノム編集を必要とする追加の研究に使用することができ、より一般的には、任意の昆虫種にCRISPR / Cas9ゲノム編集を採用することに関連しています。

<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蚊は、西ナイルウイルスやイヌの心臓虫やゾウガシスなどのフィラリア線虫によって引き起こされる病気を含む人間と動物の健康に悪影響を及ぼすいくつかの疾患の主要なベクターです。最近では、CRISPR/Cas9ゲノム編集は、アノフェレス属に属する蚊を含むいくつかの昆虫種の新たに敷設された胚にガイドRNA(gRNA)と複合体化されたCas9タンパク質を注入することによって、部位指向の突然変異を誘導するために使用されている。Culex蚊の操作と注入は、これらの蚊が他の種の蚊のように個別に卵をいかだに置くのではなく、いかだで直立させるので、少し難しいです。ここでは、gRNAを設計し、Cas9タンパク質とそれらを複雑にする方法、卵を産むためにCulexピピエンの雌の蚊を誘導する方法、およびCas9 / gRNAを用いたマイクロインジェクションのために新しく産卵した胚を調製して注入する方法について説明します。また、必要な突然変異のために注入された蚊を飼育およびスクリーニングする方法についても説明する。代表的な結果は、この技術がCulex蚊のゲノムに部位指向突然変異を誘導するために使用され、わずかな改変を伴って、他の蚊種でもヌル突然変異体を生成するために使用できることを示している。

記載されたプロトコルを用いて、Cx.pipiensの胚を正常に注入することができ、注入された胚の間で高い生存率を観察した(〜55%、図1)。以前の試験では、卵胞の前部が医療ドレッシングストリップに取り付けられ、蚊の幼虫が絨毛膜から脱出するのを防ぎ、水に泳ぐことに成功したため、生存率が低かった。前端が医療用ドレッシングのストリップを超えて延びるこ...

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は、非モデル生物の遺伝子機能を特徴付けるためにますます使用されています。このプロトコルは、注射ミックスの調製から蚊の胚の取得と注入に、および必要な突然変異のために注入された蚊とその子孫を飼育、横断、およびスクリーニングする方法に 、Culex pipiensのノックアウトラインを生成する方法を記述します。

CRISPR/Cas9を使用してヌル突然変異を生成するための Culex 蚊の胚の調製と注入

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

preparing-injecting-embryos-culex-mosquitoes-to-generate-null

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

6.8K ビュー.  The Ohio State University. CRISPR/Cas9は、非モデル生物の遺伝子機能を特徴付けるためにますます使用されています。このプロトコルは、注射ミックスの調製から蚊の胚の取得と注入に、および必要な突然変異のために注入された蚊とその子孫を飼育、横断、およびスクリーニングする方法に 、Culex pipiensのノックアウトラインを生成する方法を記述します。

ビデオ: 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.

ハイスループットフォーマットでの遺伝子型CRISPR / Cas9媒介ノックアウト変異体に蛍光PCR-キャピラリーゲル電気泳動法を用いました

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

遺伝学

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.

迅速かつ安易なパイプラインを生成する突然変異点ゲノムにc. の elegansを用いた CRISPR/Cas9 Ribonucleoproteins

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.

ヒト多能性幹細胞におけるCRISPR-CAS9を用いて定義されたゲノム修飾の生成

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.

キュレックス・キンケファシアトゥスにおける効率的な部位特異的変異誘発のための胚微小注入技術

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.

CRISPR/Cas9突然変異誘発のためのサンドフライ(フレボトムスパパタシ)胚微小注射

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.

遺伝子組み換えアノフェリン蚊の小さいケージ実験室の試験

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.

遺伝子組み換えアノフェリン蚊集団におけるインデル変異を検出するデジタル液滴PCR

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.

モスキートAedes aegyptiにおける高解像度メルト分析を用いたCRISPR/Cas9突然変異誘発に続くインデル検出

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.

CRISPR-Cas9を用いた黄熱病ネ ッタイシマカ のゲノム編集

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.

CRISPR/Cas9技術を用いた渡りバッタのホモ接合変異体の構築

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 ...

CRISPR/Cas9を使用してヌル突然変異を生成するための Culex 蚊の胚の調製と注入

要約

さらに動画を探す

この動画の章

ハイスループットフォーマットでの遺伝子型CRISPR / Cas9媒介ノックアウト変異体に蛍光PCR-キャピラリーゲル電気泳動法を用いました

迅速かつ安易なパイプラインを生成する突然変異点ゲノムにc. の elegansを用いた CRISPR/Cas9 Ribonucleoproteins

ヒト多能性幹細胞におけるCRISPR-CAS9を用いて定義されたゲノム修飾の生成

キュレックス・キンケファシアトゥスにおける効率的な部位特異的変異誘発のための胚微小注入技術

CRISPR/Cas9突然変異誘発のためのサンドフライ(フレボトムスパパタシ)胚微小注射

遺伝子組み換えアノフェリン蚊の小さいケージ実験室の試験

遺伝子組み換えアノフェリン蚊集団におけるインデル変異を検出するデジタル液滴PCR

モスキートAedes aegyptiにおける高解像度メルト分析を用いたCRISPR/Cas9突然変異誘発に続くインデル検出

CRISPR-Cas9を用いた黄熱病ネッタイシマカのゲノム編集

CRISPR/Cas9技術を用いた渡りバッタのホモ接合変異体の構築

CRISPR/Cas9を使用してヌル突然変異を生成するための Culex 蚊の胚の調製と注入

要約

さらに動画を探す

この動画の章

ハイスループットフォーマットでの遺伝子型CRISPR / Cas9媒介ノックアウト変異体に蛍光PCR-キャピラリーゲル電気泳動法を用いました

迅速かつ安易なパイプラインを生成する突然変異点ゲノムにc. の elegansを用いた CRISPR/Cas9 Ribonucleoproteins

ヒト多能性幹細胞におけるCRISPR-CAS9を用いて定義されたゲノム修飾の生成

キュレックス・キンケファシアトゥスにおける効率的な部位特異的変異誘発のための胚微小注入技術

CRISPR/Cas9突然変異誘発のためのサンドフライ(フレボトムスパパタシ)胚微小注射

遺伝子組み換えアノフェリン蚊の小さいケージ実験室の試験

遺伝子組み換えアノフェリン蚊集団におけるインデル変異を検出するデジタル液滴PCR

モスキートAedes aegyptiにおける高解像度メルト分析を用いたCRISPR/Cas9突然変異誘発に続くインデル検出

CRISPR-Cas9を用いた黄熱病ネ ッタイシマカ のゲノム編集

CRISPR/Cas9技術を用いた渡りバッタのホモ接合変異体の構築

CRISPR-Cas9を用いた黄熱病ネッタイシマカのゲノム編集