Ringraziamo il Dr. David O'Brochta e tutti i membri della Rete di ricerca di coordinamento delle tecnologie genetiche degli insetti per l'aiuto e la formazione che forniscono a noi e ad altri sull'implementazione delle tecnologie genetiche. Ringraziamo in particolare Channa Aluvihare per aver ottimizzato il protocollo di micromanipolazione per consentire l'iniezione e la schiusa degli embrioni Culex. Ringraziamo anche Devante Simmons e Joseph Urso, studenti universitari che lavorano nel laboratorio Meuti, per la loro assistenza nella cura e nello screening delle zanzare transgeniche, e Zora Elmkami dell'ITF per l'assistenza all'allevamento e la preparazione delle zanzare per l'iniezione. Questo lavoro è stato supportato da una sovvenzione interdisciplinare Seeds dell'Istituto di malattie infettive dell'OSU fornita a MEM.

<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 lavora per l'Insect Transformation Facility, che fornisce servizi di modificazione genetica degli insetti.

Questo protocollo presenta metodi per introdurre mutazioni specifiche nel genoma delle zanzare Culex e può essere utilizzato anche per modificare il genoma di altre zanzare. Il protocollo è significativo in quanto fornisce dettagli specifici non solo su come preparare i materiali di iniezione, ma anche una panoramica video dettagliata su come indurre le zanzare a dedurre le uova, nonché su come preparare e iniettare quelle uova. Riassumiamo anche come sfruttare la biologia della femmina Cx. pipiens p...

Le zanzare Culex sono distribuite in tutte le regioni temperate e tropicali del mondo e trasmettono diversi virus mortali tra cui il virus del Nilo occidentale1, l'encefalitedi St. Louis2 e i nematodi filariali che causano la filaria canina3 e l'elefantiasi4. I membri del complesso Culex pipiens, che comprende Cx. quinquefasciatus, Cx. pipiens pipiens e Cx. pipiens molestus, mostrano variazioni sorprendenti in molti aspetti della loro biologia. Ad esempio, mentre Cx. quinquefasciatus e Cx. pipiens molestus non sono in grado di entrare in una dormienza svernante5,6, Cx. pipiens pipiens mostrano robuste risposte stagionali ed entrano in diapausa in risposta ai giorni brevi7,8. Inoltre, Cx. pipiens molestus tendono ad essere più antropofili mentre Cx. pipiens e Cx. quinquefasciatus sono più zoofili6. Tuttavia, negli Stati Uniti e in molti altri luoghi del mondo, queste specie si incrociano, il che ha forti implicazioni per la trasmissione della malattia come ibridi di Cx. pipiens pipiens e Cx. pipiens molestus sono alimentatori opportunistici e morderanno sia gli uccelli che gliesseriumani 9 , fungendo così da vettori ponte per il virus del Nilo occidentale. Lo studio di questi e altri aspetti affascinanti della biologia delle zanzare Culex è stato ostacolato, in parte, perché le zanzare Culex sono leggermente più difficili da allevare in laboratorio rispetto alle zanzare Aedes, che producono uova quiescenti e resistenti all'essiccazione10 e perché gli strumenti molecolari funzionali non sono così ben sviluppati per le specie Culex. 
L'editing del genoma CRISPR/Cas9 è una potente tecnologia che è stata utilizzata per valutare la biologia di diverse importanti specie di zanzare11,12,13,tra cui la zanzara della casa meridionale, Culex quinquefasciatus14,15,16. Questa tecnologia, sviluppata da Jennifer Doudna ed Emmanuelle Charpentier, sfrutta una difesa batterica naturale contro i virus da parte di endonucleasi associate a CRISPR di derivazione batterica (proteine Cas; vedi recensione di Van der Oost et al.17). Quando iniettate in embrioni animali, le proteine Cas9 in combinazione con un RNA guida appropriato possono produrre rotture a doppio filamento all'interno del genoma. Questo viene fatto più frequentemente utilizzando la proteina Cas9 che è complessata con RNA guida, che dirige l'attività dell'endonucleasi in una regione specifica del genoma. Dopo che la proteina Cas9 ha creato una rottura a doppio filamento site-specific, il macchinario cellulare tenta di riparare la rottura utilizzando uno dei due meccanismi. Il primo comporta la legatura delle due estremità insieme attraverso l'unione finale non omologa (NHEJ), che è soggetta a errori e spesso produce inserzioni e delezioni out-frame nel genoma che possono portare a proteine non funzionali, generando così una mutazione knock-out. In alternativa, il macchinario cellulare potrebbe utilizzare la riparazione diretta dall'omologia (HDR) trovando sequenze simili per riparare correttamente la rottura. La sequenza simile può essere fornita dal secondo cromosoma all'interno dell'organismo (vedi revisione18). Tuttavia, se la sequenza riparata corrisponde esattamente alla sequenza originale, la proteina Cas9 sarà in grado di tagliare nuovamente il DNA. In alternativa, i ricercatori possono anche includere un plasmide donatore che contiene sequenze omologhe su entrambi i lati del sito di taglio della sequenza target con una sequenza di riparazione alternativa - spesso una proteina marcatore fluorescente, una versione modificata del gene originale o altre modifiche - che possono essere copiate e inserite nel genoma o "knocked-in".
Il tempismo è fondamentale quando si iniettano embrioni, e questo è particolarmente vero quando si utilizza l'editing del genoma CRISPR / Cas9 per creare mutazioni negli insetti. Questo perché la proteina Cas9 e i gRNA hanno la maggiore capacità di generare mutazioni solo quando l'embrione è nel suo stato sinciziale, prima che si siano formate membrane cellulari e quando più nuclei siano accessibili all'interno dell'embrione. Per le zanzare, i nuclei raggiungono la periferia ~ 2-4 ore dopo l'ovodeposizione, a seconda della temperatura19, e quindi la microiniezione di successo deve avvenire prima di questo momento. Inoltre, la proteina Cas9 taglierà qualsiasi DNA nucleare a cui può accedere, in modo tale che l'individuo risultante dall'iniezione conterrà un mosaico di cellule, alcune con la mutazione desiderata e altre no. Affinché queste mutazioni siano ereditate con successo, la proteina Cas9 deve tagliare il DNA che risiede nella linea germinale che darà origine alle future uova e spermatozoi. Per garantire che le mutazioni siano generate nella linea germinale è meglio iniettare tutti i materiali vicino alla posizione delle cellule polari all'interno dell'embrione, che sono i progenitori della linea germinale dell'insetto. Le cellule polari si trovano vicino all'estremità posteriore degli embrioni di Culex 20. Oltre a iniettare embrioni, è imperativo sviluppare un piano attento per l'incrocio e lo screening della prole al fine di rilevare la mutazione desiderata.
Questo protocollo descrive come generare gRNA e complessarli con la proteina Cas9 per preparare miscele di iniezione, nonché come indurre le zanzare femmine di Culex pipiens a depostare le uova e come preparare e iniettare quelle uova per l'editing del genoma mediato da CRISPR / Cas9. Inoltre, descriviamo come allevare, incrociare e schermare gli embrioni iniettati e la loro progenie per confermare che la mutazione desiderata è stata ottenuta. Usando questo protocollo, abbiamo generato mutazioni nulle per un gene di interesse, ciclo,nel ceppo Buckeye di Culex pipiens. Questo ceppo è stato originariamente istituito nel 2013 da zanzare raccolte sul campo a Columbus, Ohio ed è mantenuto dal laboratorio Meuti. Questo protocollo può essere utilizzato per ulteriori studi che richiedono l'editing del genoma CRISPR / Cas9 nelle zanzare Culex, così come in altre specie di zanzare e, più in generale, è rilevante per l'impiego dell'editing del genoma CRISPR / Cas9 a qualsiasi specie di insetti.

<table border="0" cellpadding="0" cellspacing="0" style="width:622px;" width="623">
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	</colgroup>
	<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

Le zanzare Culex sono i principali vettori di diverse malattie che hanno un impatto negativo sulla salute umana e animale, tra cui il virus del Nilo occidentale e le malattie causate da nematodi filariali come la filaria canina e l'elefantiasi. Recentemente, l'editing del genoma CRISPR / Cas9 è stato utilizzato per indurre mutazioni dirette al sito iniettando una proteina Cas9 che è stata complessata con un RNA guida (gRNA) in embrioni appena deposti di diverse specie di insetti, comprese le zanzare che appartengono ai generi Anopheles e Aedes. Manipolare e iniettare zanzare Culex è leggermente più difficile in quanto queste zanzare depongono le uova in posizione verticale in zattere piuttosto che individualmente come altre specie di zanzare. Qui descriviamo come progettare gRNA, complessarli con la proteina Cas9, indurre le zanzare femmine di Culex pipiens a depostare le uova e come preparare e iniettare embrioni appena deposti per la microiniezione con Cas9 / gRNA. Descriviamo anche come allevare e schermare le zanzare iniettate per la mutazione desiderata. I risultati rappresentativi dimostrano che questa tecnica può essere utilizzata per indurre mutazioni site-directed nel genoma delle zanzare Culex e, con lievi modifiche, può essere utilizzata per generare mutanti nulli anche in altre specie di zanzare.

Utilizzando il protocollo descritto, siamo stati in grado di iniettare con successo embrioni di Cx. pipiense abbiamo osservato un alto tasso di sopravvivenza tra gli embrioni iniettati (~ 55%, Figura 1). Gli studi precedenti avevano una percentuale inferiore di sopravvivenza, probabilmente perché l'anteriore del follicolo dell'uovo era attaccato alla striscia medicazione, impedendo alle larve di zanzara di sfuggire al coro e nuotare con successo nell'acqua. Garantire che l'estremit...

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 è sempre più utilizzato per caratterizzare la funzione genica in organismi non modello. Questo protocollo descrive come generare linee knock-out di Culex pipiens, dalla preparazione di miscele di iniezione, all'ottenimento e all'iniezione di embrioni di zanzara, nonché come allevare, incrociare e schermare le zanzare iniettate e la loro progenie per le mutazioni desiderate.

Preparazione e iniezione di embrioni di zanzare Culex per generare mutazioni nulle utilizzando CRISPR / Cas9

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 Views.  The Ohio State University. CRISPR/Cas9 è sempre più utilizzato per caratterizzare la funzione genica in organismi non modello. Questo protocollo descrive come generare linee knock-out di Culex pipiens, dalla preparazione di miscele di iniezione, all'ottenimento e all'iniezione di embrioni di zanzara, nonché come allevare, incrociare e schermare le zanzare iniettate e la loro progenie per le mutazioni desiderate.

Video: 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.

Iniezione di An. Gli embrioni stephensi per generare la malaria resistenti zanzare

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

Ricerca

Biologia

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.

Microiniezione di A. aegypti embrioni per ottenere zanzare transgeniche

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.

Raccolta e Preparazione Drosophila Gli embrioni per la registrazione elettrofisiologici e altre procedure

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.

Generazione di Genomic eliminazioni in linee cellulari di mammiferi via CRISPR / Cas9

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.

Silenziamento della scintilla: 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 ...

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.

Ingegneria genomica delle cellule B umane primarie utilizzando 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 ...

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 endoteliale mediato a transizione mesenchimale (EndMT) e la valutazione funzionale degli effettori EndMT utilizzando 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 ...

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.

Preparazione di zanzare Aedes aegypti maschi irradiate e marcate per il rilascio in un programma operativo di tecnica degli insetti sterili

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.

Costruzione di mutanti omozigoti di locuste migratorie utilizzando la tecnologia CRISPR/Cas9

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.