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>
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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. 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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 border="0" cellpadding="0" cellspacing="0" style="width:622px;" width="623">
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	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Artificial Membrane Feeder</td>
			<td style="width:121px;">Hemotek</td>
			<td style="width:119px;">SP5W1-3</td>
			<td style="width:167px;">Company location: Blackburn, UK</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">ATP</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:119px;">18330019</td>
			<td style="width:167px;">Company location: Carlsbad, CA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Borosilicate glass mirocapillary tubes, 1 mm outer diameter</td>
			<td style="width:121px;">World Precision Instruments</td>
			<td style="width:119px;">1B100-6</td>
			<td style="width:167px;">Company Location: Sarasota, FL, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">BV10 Needle Beveler</td>
			<td style="width:121px;">Sutter Instruments</td>
			<td style="width:119px;">BV-10-B</td>
			<td style="width:167px;">Company Location: Nobato, CA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Whatman Circular filter paper (12.5 cm)</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">WHA1001125</td>
			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Conical tube (50 mL)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">339652</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Fisherbrand course filter paper with fast flow rate</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">09-800</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Cover glass (24 x 40 mm)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-311-20</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Dental dam</td>
			<td style="width:121px;">Henry Schein Inc</td>
			<td style="width:119px;">1010171</td>
			<td style="width:167px;">Company Location: Melville, NY USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Scotch double-sided tape</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">NC0879005</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">FemtoJet 4i microinjector</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:119px;">5252000021</td>
			<td style="width:167px;">Company Location: Hamburg, Germany</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Glass vial (2 dram)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">033401C</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Halocarbon oil</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">H8898-50ML</td>
			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">P-2000 Laser Needle Puller</td>
			<td style="width:121px;">Sutter Instruments</td>
			<td style="width:119px;">P-2000/G</td>
			<td style="width:167px;">Company Location: Nobato, CA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Parafilm</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-998-944</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">PC-100 Weighted Needle Puller</td>
			<td style="width:121px;">Narishige</td>
			<td style="width:119px;">PC-100</td>
			<td style="width:167px;">This is compatabile with the earlier PC-10 model, which has been discontinued. Company Location: Amityville, NY, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Phire Direct PCR Kit</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F140WH</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Kodak Photo-Flo (1%)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-268-05</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Quartz glass mirocapillary tubes, 1 mm outer diameter</td>
			<td style="width:121px;">Capillary Tube Supplies Limited</td>
			<td style="width:119px;">QGCT 1.0</td>
			<td style="width:167px;">Company Location: Cornwall, UK</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Guide-it&#8482; sgRNA Screening Kit</td>
			<td style="width:121px;">Takara, Bio USA</td>
			<td style="width:119px;">632639</td>
			<td style="width:167px;">This kit allows you to determine if gRNAs cut DNA sequences in vitro. Company Location: Mountain View, CA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Sigmacote</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">SL2-100ML</td>
			<td style="width:167px;">Company Location: St. Louis, MO, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Small petri dishes (35X10 mm)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">50-190-0273</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Sodium citrate chicken blood</td>
			<td style="width:121px;">Lampire biologicals</td>
			<td style="width:119px;">7201406</td>
			<td style="width:167px;">Company Location: Everett, PA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Fisherbrand Square petri dish (10 cm x 10 cm)</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">FB0875711A</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Tegaderm</td>
			<td style="width:121px;">Henry Schein Inc.</td>
			<td style="width:119px;">7771180</td>
			<td style="width:167px;">Company Location: Melville, NY USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Tropical fish food</td>
			<td style="width:121px;">Tetramin</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Whatman filter paper</td>
			<td style="width:121px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">09-927-826</td>
			<td style="width:167px;">Company Location: Waltham, MA, USA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Whatman filter paper, 4.25 cm</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:119px;">1001-042</td>
			<td style="width:167px;">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

Research

JoVE Journal

Biology

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

6.7K 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

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

の注入。ハマダラカの胚は、マラリア耐性の蚊を生成する

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

生物学

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

トランスジェニック蚊を取得するA.ネッタイシマカの胚のマイクロインジェクション

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

収穫と準備ショウジョウバエ</em電気生理学的記録とその他の手続のための>胚

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

CRISPR / Cas9経由哺乳動物細胞株におけるゲノム欠失の生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

火花のサイレンシング:弱い電気魚のCRISPR/Cas9ゲノム編集

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

CRISPR/Cas9を用いた初等ヒトB細胞のゲノム工学

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

TGF β媒介性間葉転移(EndMT)およびCRISPR/Cas9遺伝子編集を用いたEndMTエフェクターの機能評価

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no prevention. Control of mosquito vectors can rely on chemicals applied to the immature and adult stages, which can contribute to the mortality of non-targets and more importantly, lead to insecticide resistance in the vector. The sterile insect technique (SIT) is a method of controlling populations of pests through the release of sterilized adult males that mate with wild females to produce non-viable offspring. This paper describes the process of producing sterile males for use in an operational SIT program for the control of Aedes aegypti mosquitoes. Outlined here are the steps used in the program including rearing and maintaining a colony, separating male and female pupae, irradiating and marking adult males, and shipping Aedes aegypti males to the release site. Also discussed are procedural caveats, program limitations, and future objectives.

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The sterile insect technique (SIT) is used to control specific, medically important mosquito populations that may be resistant to chemical controls. Here, we describe a method of mass rearing and preparation of sterile male mosquitoes for release in an operational SIT program targeting the Aedes aegypti mosquito.

照射され、マークされたオスの ネッタイシマカ を、操作可能な滅菌昆虫技術プログラムで放出するための準備

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no ...

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 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

Methods Article

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