Name: construction of homozygous mutants of migratory locust using crispr/cas9 technology
Uploaded: 2022-03-16T15:00:00
Description: Watch this Scientific Journal Video about Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology at JoVE.com

This work was supported by the National Natural Science Foundation of China (32070502, 31601697, 32072419 and the China Postdoctoral Science Foundation (2020M672205).

1. Target site selection and sgRNA design
<ol>
	<li>Collect as much sequence information as possible for the gene of interest through literature research and/or searching for the mRNA or coding DNA sequence (CDS) of the gene at NCBI and locust database24.</li>
	<li>Compare the sequence of the interested gene to its genomic DNA sequence to distinguish the exon and intron regions.</li>
	<li>Select a candidate region for target site design based on the research purpose. Design prim...

<ol>
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(Orthoptera : Acrididae).</a> International Journal of Insect Morphology and Embryology. 14 (6), 361-379 (1985).</li><li>Barry, S. 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=Injecting+Gryllus+bimaculatus+eggs.">Injecting Gryllus bimaculatus eggs.</a> Journal of Visualized Experiments: JoVE. (150), e59726 (2019).</li><li>Watanabe, T., Noji, S., Mito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+the+cricket,+Gryllus+bimaculatus.">Genome editing in the cricket, Gryllus bimaculatus.</a> Methods in Molecular Biology. 1630, 219-233 (2017).</li><li>Du, M. H., 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=Suppression+of+Laccase+2+severely+impairs+cuticle+tanning+and+pathogen+resistance+during+the+pupal+metamorphosis+of+Anopheles+sinensis+(Diptera:+Culicidae).">Suppression of Laccase 2 severely impairs cuticle tanning and pathogen resistance during the pupal metamorphosis of Anopheles sinensis (Diptera: Culicidae).</a> Parasites &amp; Vectors. 10 (1), 171 (2017).</li><li>Eisner, T., Shepherd, J., Happ, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tanning+of+grasshopper+eggs+by+an+exocrine+secretion.">Tanning of grasshopper eggs by an exocrine secretion.</a> Science. 152 (3718), 95-97 (1966).</li><li>Ho, K., Dunin-Borkowski, O. 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Locusts have been among the most devastating pests to agriculture since the civilization of human beings23. CRISPR/Cas9-based genome editing technology is a powerful tool for providing better knowledge of the biological mechanisms in locusts as well as a promising pest control strategy. Thus, it is of great benefit to develop an efficient and easy-to-use method of CRISPR/Cas9-mediated construction of homozygous locust mutants. Although some great works have been reported and provided some basic wo...

Gene editing technologies could be used to introduce insertions or deletions into a specific genome locus to artificially modify the target gene on purpose1. In the past years, CRISPR/Cas9 technology has developed rapidly and has a growing scope of applications in various fields of life sciences2,3,4,5,6. The CRISPR/Cas9 system was discovered back in 19877, and widely found in bacteria and archaea. Further research indicated that it was a prokaryotic adaptive immune system that depends on the RNA-guided nuclease Cas9 to fight against phages8. The artificially modified CRISPR/Cas9 system mainly consists of two components, a single guide RNA (sgRNA) and the Cas9 protein. The sgRNA is made up of a CRISPR RNA (crRNA) complementary to the target sequence and an auxiliary trans-activating crRNA (tracrRNA), which is relatively conserved. When the CRISPR/Cas9 system is activated, the sgRNA forms a ribonucleoprotein (RNP) with the Cas9 protein and guides Cas9 to its target site via the base pairing of RNA-DNA interactions. Then, the double-strand DNA can be cleaved by the Cas9 protein and as a result, the double-strand break (DSB) emerges near the protospacer adjacent motif (PAM) of the target site9,10,11,12. To mitigate the damage caused by the DSBs, cells would activate comprehensive DNA damage responses to efficiently detect the genomic damages and initiate the repair procedure. There are two distinct repair mechanisms in the cell: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is the most common repair pathway that can repair DNA double-strand breaks quickly and prevents cell apoptosis. However, it is error-prone because of leaving small fragments of insertions and deletions (indels) near the DSBs, which usually results in an open reading frame shift and thus can lead to gene knock-out. In contrast, homologous repairment is quite a rare event. On the condition that there is a repair template with sequences homologous to the context of the DSB, cells would occasionally repair the genomic break according to the nearby template. The result of HDR is that the DSB is precisely repaired. Especially, if there is an additional sequence between the homologous sequences in the template, they would be integrated into the genome through HDR, and in this way, the specific gene insertions could be realized13.
With the optimization and development of the sgRNA structures and Cas9 protein variants, the CRISPR/Cas9-based genetic editing system has also been successfully applied in research of insects, including but not limited to Drosophila melanogaster, Aedes aegypti, Bombyx mori, Helicoverpa Armigera, Plutella xylostella, and Locusta migratoria14,15,16,17,18,19. To the best of the authors&#39; knowledge, although RNPs consisting of the Cas9 protein and in vitro transcribed sgRNA have been used for locust genome editing20,21,22, a systematic and detailed protocol for CRISPR/Cas9 ribonuclease mediated construction of homozygous mutants of the migratory locust is still lacking.
The migratory locust is an important agricultural pest that has a global distribution and poses substantial threats to food production, being especially harmful to gramineous plants, such as wheat, maize, rice, and millet23. Gene function analysis based on genome editing technologies can provide novel targets and new strategies for the control of migratory locusts. This study proposes a detailed method for knocking out migratory locust genes via the CRISPR/Cas9 system, including the selection of target sites and design of sgRNAs, in vitro synthesis and verification of the sgRNAs, microinjection and culture of eggs, estimation of mutation rate at the embryonic stage, detection of mutants as well as passage and preservation of the mutants. This protocol could be used as a basal reference for manipulation of the vast majority of locust genes and can provide valuable references for genome editing of other insects.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10&#215;NEBuffer r3.1</td>
			<td>New England Biolabs</td>
			<td>B7030S</td>
			<td>&#160;The buffer of &#160;in vitro Cas9 cleavage assays</td>
		</tr>
		<tr>
			<td>2xEs Taq MasterMix (Dye)</td>
			<td>Cwbio</td>
			<td>CW0690</td>
			<td>For gene amplification</td>
		</tr>
		<tr>
			<td>2xPfu MasterMix (Dye)</td>
			<td>Cwbio</td>
			<td>CW0686</td>
			<td>For gene amplification</td>
		</tr>
		<tr>
			<td>CHOPCHOP</td>
			<td></td>
			<td></td>
			<td>Online website for designing sgRNAs, http://chopchop.cbu.uib.no/.</td>
		</tr>
		<tr>
			<td>CRISPOR</td>
			<td></td>
			<td></td>
			<td>Online website for designing sgRNAs, http://crispor.org.</td>
		</tr>
		<tr>
			<td>CRISPRdirect</td>
			<td></td>
			<td></td>
			<td>Online website for designing sgRNAs, http://crispr.dbcls.jp/.</td>
		</tr>
		<tr>
			<td>Electrophoresis power supply</td>
			<td>LIUYI BIOLOGY</td>
			<td>DYY-6D</td>
			<td>Separation of nucleic acid molecules of different sizes</td>
		</tr>
		<tr>
			<td>Eppendorf Tube</td>
			<td>Eppendorf</td>
			<td>30125177</td>
			<td>For sample collection, etc.</td>
		</tr>
		<tr>
			<td>Fine brushes</td>
			<td>Annigoni</td>
			<td>1235</td>
			<td>For cleaning and isolating eggs. Purchased online.</td>
		</tr>
		<tr>
			<td>Flaming/brown micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P97</td>
			<td>For making the microinjection needles</td>
		</tr>
		<tr>
			<td>Gel Extraction Kit</td>
			<td>Cwbio</td>
			<td>CW2302</td>
			<td>DNA recovery and purification</td>
		</tr>
		<tr>
			<td>Gel Imaging Analysis System</td>
			<td>OLYMPUS</td>
			<td>Gel Doc XR</td>
			<td>Observe the electrophoresis results</td>
		</tr>
		<tr>
			<td>GeneTouch Plus</td>
			<td>Bioer</td>
			<td>B-48DA</td>
			<td>For gene amplification</td>
		</tr>
		<tr>
			<td>Glass electrode capillary</td>
			<td>Gairdner</td>
			<td>GD-102</td>
			<td>For making&#160;injection needles with&#160;a&#160;micropipette Puller</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>MEMMERT</td>
			<td>INplus55</td>
			<td>For migratory locust embryo culture</td>
		</tr>
		<tr>
			<td>Metal bath</td>
			<td>TIANGEN</td>
			<td>AJ-800</td>
			<td>For heating the sample</td>
		</tr>
		<tr>
			<td>Micro autoinjector</td>
			<td>Eppendorf</td>
			<td>5253000068</td>
			<td>Microinjection of embryos early in development</td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td>Allsheng</td>
			<td>MTV-1</td>
			<td>Used for mixing reagents</td>
		</tr>
		<tr>
			<td>Microgrinder</td>
			<td>NARISHIGE</td>
			<td>EG-401</td>
			<td>To ground the tip of injection needle</td>
		</tr>
		<tr>
			<td>Microloader</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>For loading solutions into the injection needles.</td>
		</tr>
		<tr>
			<td>Micromanipulation system</td>
			<td>Eppendorf</td>
			<td>TransferMan 4r</td>
			<td>An altinative manipulation system for microinjection</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>cnoptec</td>
			<td>SZ780</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>Motor-drive Manipulator</td>
			<td>NARISHIGE</td>
			<td>MM-94</td>
			<td>For controling the position of the micropipette during the microinjection precedure</td>
		</tr>
		<tr>
			<td>Multi-Sample Tissue Grinder</td>
			<td>jingxin</td>
			<td>Tissuelyser-64</td>
			<td>Grind and homogenize the eggs</td>
		</tr>
		<tr>
			<td>ovipisition pot</td>
			<td>ChangShengYuanYi</td>
			<td>CS-11</td>
			<td>Filled with wet sterile sand for locust ovipositing in it. The oocysts are collected from this container. Purchased online.</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>ParafilmM</td>
			<td>PM996</td>
			<td>For wrapping the petri dishes.</td>
		</tr>
		<tr>
			<td>pEASY-T3 Cloning Kit</td>
			<td>TransGen Biotech</td>
			<td>CT301-01</td>
			<td>For TA cloning</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>NEST</td>
			<td>752001</td>
			<td>For culture and preservation of&#160; the eggs.</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Eppendorf</td>
			<td>Research&#174;plus</td>
			<td>&#160;For sample loading</td>
		</tr>
		<tr>
			<td>plastic culture cup</td>
			<td></td>
			<td></td>
			<td>For rearing locusts seperately and any plastic cup big enough (not less than 1000 mL in volume) will do. Purchased online.</td>
		</tr>
		<tr>
			<td>Precision gRNA Synthesis Kit</td>
			<td>Thermo</td>
			<td>A29377</td>
			<td>For sgRNA synthesis</td>
		</tr>
		<tr>
			<td>Primer Premier</td>
			<td>PREMIER Biosoft</td>
			<td>Primer Premier 5.00</td>
			<td>For primer design</td>
		</tr>
		<tr>
			<td>SnapGene</td>
			<td>Insightful Science</td>
			<td>SnapGene&#174;4.2.4</td>
			<td>For analyzing&#160; sequences</td>
		</tr>
		<tr>
			<td>Steel balls</td>
			<td>HuaXinGangQiu</td>
			<td>HXGQ60</td>
			<td>For sample grinding.Purchased online.</td>
		</tr>
		<tr>
			<td>Tips</td>
			<td>bioleaf</td>
			<td>D781349</td>
			<td>&#160;For sample loading</td>
		</tr>
		<tr>
			<td>Trans DNA Marker II</td>
			<td>TransGen Biotech</td>
			<td>BM411-01</td>
			<td>Used to determine gene size</td>
		</tr>
		<tr>
			<td>TrueCut Cas9 Protein v2</td>
			<td>Thermo</td>
			<td>A36496</td>
			<td>Cas9 protein</td>
		</tr>
		<tr>
			<td>UniversalGen DNA Kit</td>
			<td>Cwbio</td>
			<td>CWY004</td>
			<td>For genomic DNA extraction</td>
		</tr>
		<tr>
			<td>VANNAS Scissors</td>
			<td>Electron Microscopy Sciences</td>
			<td>72932-01</td>
			<td>For cutting off the antennae</td>
		</tr>
		<tr>
			<td>Wheat</td>
			<td></td>
			<td></td>
			<td>To generate wheat seedlings as the food for locusts. Bought from local farmers.</td>
		</tr>
		<tr>
			<td>ZiFiT</td>
			<td></td>
			<td></td>
			<td>Online website for designing sgRNAs, http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx.</td>
		</tr>
	</tbody>
</table>

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 protocol contains the detailed steps for generating homozygous mutants of the migratory locusts with the RNP consisting of Cas9 protein and in vitro synthesized sgRNA. The following are some representative results of CRISPR/Cas9-mediated target gene knockout in locusts, including target selection, sgRNA&#160;synthesis and verification (Figure 1A), egg collection and injection, mutant screening and passaging, cryopreservation, and resuscitation of the homozygous eggs.
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Watch this Scientific Journal Video about Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology at JoVE.com

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

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.

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 ...

Le criquet migrateur est un test agricole important. Cette méthode fournit un protocole efficace et facile à céder pour les méthodes de pression génique de l’Il s’agit d’une méthode systématique et détaillée de construction CRISPR/CAS9 de mutants homozygotes du criquet, y compris la collecte d’œufs, le criblage des mutants et le maintien homozygote. Pour commencer, préparez les aiguilles d’injection en tirant les capillaires en verre avec un extracteur de micro-pipettes.Réglez les paramètres comme décrit dans le manuscrit textuel, puis broyez la pointe de l’aiguille d’injection avec une micro-meuleuse. Préparer la protéine ribonucléo pour l’injection en mélangeant un microlitre de protéine Cas9 avec un microlitre de l’ARN guide unique vérifié. Ajoutez ensuite huit microlitres d’eau stérile libre de l’ARN pour obtenir le volume final du mélange, 10 microlitres.Bien mélanger la solution et la placer sur de la glace. Ensuite, récupérez les gousses d’œufs fraîchement pondues de la gousse de position des ovules et attendez le bronzage des œufs. Après 30 minutes, utilisez une brosse fine pour isoler les œufs des gousses dans l’eau et lavez les œufs trois fois avec de l’eau stérile.Ensuite, placez les œufs dans une boîte de Petri et ajoutez de l’eau stérile pour les garder humides. Remplissez l’aiguille d’injection avec la solution de protéine ribonucléo préparée et chargez l’injection sur le micromanipulateur. Définissez les paramètres de micro-injection sur 300 hectopascals de pression d’injection, 0,5 seconde de temps d’injection et 25 hectopascals de pression de compensation.Lorsque les paramètres sont réglés, appuyez sur la pédale pour évaluer le volume de la solution injectée. Ajustez la pression et le temps d’injection pour vous assurer que le volume d’injection est d’environ 50 à 100 nanolitres. Après avoir disposé régulièrement les œufs stériles sur le tampon d’injection, placez le tampon sur la table d’objets du microscope et ajustez le grossissement du microscope jusqu’à ce que les œufs soient observés.Ajustez ensuite l’aiguille de micro-injection au microscope jusqu’à ce que l’embout d’injection soit visible et positionnez-le près de l’œuf à injecter. Commencez l’injection à une hauteur appropriée et à un angle de 30 à 45 degrés. Insérez la pointe dans l’œuf près des micro-piles de l’œuf et appuyez sur la pédale pour accomplir l’injection.Ensuite, rétractez rapidement l’aiguille et déplacez le tampon d’injection pour l’injection de l’œuf suivant. Transférer les œufs injectés dans une boîte de culture avec un morceau de papier filtre humide. Placez-les dans un incubateur à 30 degrés Celsius.Après l’injection, vérifiez le développement des ovules injectés tous les jours. Transférer les œufs injectés dans une nouvelle boîte de culture toutes les 24 heures au cours des cinq premiers jours. Le sixième jour après l’injection, cueillez et transférez au hasard 10 œufs dans un tube et broyez correctement les œufs avec deux billes d’acier à 60 hertz pendant six minutes à l’aide d’un broyeur.Après le meulage, remettez les débris en suspension dans un millilitre de PBS. Ensuite, transférez cinq microlitres du mélange remis en suspension dans 45 microlitres d’hydroxyde de sodium à 50 millimolaires pour lyser à 95 degrés Celsius pendant cinq minutes. Pour mettre fin à la réaction de lyse alcaline, ajoutez cinq microlitres d’une solution molaire de chlorhydrate de Tris dans le système de lyse.Prenez un microlitre du produit de lyse comme modèle de PCR pour amplifier le fragment de gène cible. Après la PCR et le séquençage, comparez les résultats du séquençage avec la séquence de type sauvage pour évaluer si le système CRISPR / Cas9 a clivé le gène cible in vivo. Laisser le reste des œufs pour un développement ultérieur car les indels sont détectés au stade embryonnaire.Après le développement, le transfert et la culture, les nymphes écloses dans une cage d’élevage comme décrit dans le manuscrit. Lorsque les nymphes atteignent le cinquième stade, séparez-les avec des gobelets de culture en plastique. Effectuer des croisements à l’aide des mutants G0 et des criquets de type sauvage.Après avoir recueilli des oassists, utilisez trois à six ovules développés dans chaque oassist pour détecter les mutations comme décrit précédemment. Conservez les oassists positifs pour le développement ultérieur et abandonnez les oassists mutation-négatifs. Pour la cryoconservation, lavez les œufs avec de l’eau stérile et incuberez-les dans un plat de culture comme expliqué précédemment.Après cinq à six jours, rassemblez les œufs dans le plat de culture et recouvrez-les de fragments de papier filtre. Ensuite, enveloppez tout le plat de culture avec un film de paraffine. Placez le plat à 25 degrés Celsius pendant deux jours, suivis de deux autres jours à une température plus basse de 13 à 16 degrés Celsius.Ensuite, transférez le plat dans un réfrigérateur à six degrés Celsius. Ajouter de l’eau au plat toutes les deux semaines pour fournir un environnement humide aux œufs. Enfin, pour ressusciter les œufs cryoconservés, placez la boîte de Petri à 25 degrés Celsius pendant deux jours.Plus tard, placez les œufs dans un incubateur à 30 degrés Celsius jusqu’à l’éclosion des nymphes. Dans cette étude, le site cible du système CRISPR/Cas9 était situé dans le premier exon. Dans le test de clivage Cas9, la protéine ribonucléo CRISPR / Cas9 a pu digérer le fragment de PCR contenant le site cible avec un taux de clivage d’environ 55%Les résultats du séquençage pour l’estimation du taux de mutation au stade embryonnaire ont suggéré une édition efficace du génome sur le site cible.L’efficacité de l’édition a entraîné un taux d’éclosion des nymphes de 52,73% et 66,67% des adultes G0 étaient des mutants mosaïques. La stratégie de passage des lignées mutantes est montrée ici. Les œufs homozygotes excédentaires ont été cryoconservés pour améliorer le taux d’utilisation des homozygotes.Bien que le taux d’éclosion des œufs cryoconservés ait été réduit avec l’allongement du temps de cryoconservation, les mesures correctives telles que l’application de fragments de papier filtre et la récupération de ces œufs conservés avec un gradient de température ascendante ont été utiles pour la réanimation. Le plus important est d’essayer de limiter les dégâts mécaniques causés par la micro-injection. Cette procédure peut être utilisée comme référence pour atteindre d’autres insectes.Une partie de certains détails tels que les méthodes de collecte des œufs, d’injection et de culture devrait être modifiée. Cette technique peut également être utile pour étudier la métamorphose et l’adaptation environnementale des insectes et la construction de mutants homozygotes acridiens a accéléré les recherches dans ce domaine.

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Biology

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Recherche

Biologie

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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

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CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

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Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System

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