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 ...

The migratory locust is an important agricultural test. This method provides an efficient and easy to yield protocol for the gene pressure methods of the It is a systematic and a detailed method of a CRISPR/CAS9 construction of homozygous locust mutants, including egg collection, mutant screening and the homozygous maintaining. To begin, prepare the injection needles by pulling the glass capillaries with a micro pipette puller.Set the parameters as described in the text manuscript, then grind the tip of the injection needle with a micro grinder. Prepare ribonucleo protein for the injection by mixing one microliter of Cas9 protein with one microliter of the verified single guide RNA. Then add eight microliters of RNA's free sterile water to make the final volume of the mixture, 10 microliters.Mix the solution thoroughly and place it on ice. Next, collect the freshly laid egg pods from the ova position pod, and wait for egg tanning. After 30 minutes, use a fine brush to isolate the eggs from the egg pods in water, and wash the eggs three times with sterile water.Then place the eggs in a Petri dish and add sterile water to keep them moist. Fill the injection needle with the prepared ribonucleo protein solution, and load the injection to the micro manipulator. Set the microinjection parameters as 300 Hectopascals of injection pressure, 0.5 seconds of injection time and 25 Hectopascals of compensation pressure.When the parameters are set, press the pedal to evaluate the volume of the injected solution. Adjust the injection pressure and injection time to ensure that the injection volume is about 50 to 100 nanoliters. After arranging the sterile eggs regularly on the injection pad, place the pad on the object table of the microscope and adjust the magnification of the microscope until the eggs are observed.Then adjust the microinjection needle under the microscope until the injection tip can be seen and position the tip near the egg to be injected. Start the injection at a suitable height and an angle of 30 to 45 degrees. Insert the tip into the egg near the micro piles of the egg and press the pedal to accomplish the injection.Next, retract the needle quickly and move the injection pad for injection of the next egg. Transfer the injected eggs into a culture dish with a piece of moist filter paper. Place them in an incubator at 30 degrees Celsius.After injection, check the development of the injected eggs every day. Transfer the injected eggs to a new culture dish every 24 hours in the first five days. On the sixth day after the injection, randomly pick and transfer 10 eggs into a tube and adequately grind the eggs with two steel balls at 60 hertz for six minutes using a grinder.After grinding, resuspend the debris in one milliliter PBS. Then transfer five microliters of the resuspended mixture into 45 microliters of 50 millimolar sodium hydroxide to lyse at 95 degrees Celsius for five minutes. To terminate the alkaline lysis reaction, add five microliters of one molar Tris hydrochloride solution into the lysis system.Take one microliter of the lysis product as the PCR template to amplify the target gene fragment. After the PCR and sequencing, compare the sequencing results with the wild type sequence to evaluate whether the CRISPR/Cas9 system cleaved the target gene in vivo. Allow the rest of the eggs for subsequent development as indels are detected at the embryonic stage.After development, transfer and culture the hatched nymphs into a rearing cage as described in the manuscript. When the nymphs develop to the fifth instar stage, separate them with plastic culture cups. Perform cross-breeding using the G0 mutants and wild type locusts.After collecting oassists, use three to six developed eggs in each oassist to detect mutations as described earlier. Keep mutation positive oassists for the subsequent development and abandon the mutation negative oassists. For cryo-preservation, wash the eggs with sterile water and incubate them in a culture dish as explained before.After five to six days, gather the eggs together in the culture dish, and cover them with filter paper fragments. Then wrap the entire culture dish with a paraffin film. Place the dish at 25 degrees Celsius for two days, followed by another two days at a lower temperature of 13 to 16 degrees Celsius.Then transfer the dish to a six degree Celsius refrigerator. Add water to the dish every two weeks to provide a moist environment for the eggs. Finally, for resuscitating the cryopreserved eggs, place the Petri dish at 25 degrees Celsius for two days.Later, place the eggs in a 30 degrees Celsius incubator until the nymphs hatch. In this study, the target site for the CRISPR/Cas9 system was located in the first exon. In the Cas9 cleavage assay, the CRISPR/Cas9 ribonucleo protein could digest the PCR fragment containing the target site with a cleavage rate of about 55%The sequencing results for embryonic stage mutation rate estimation suggested efficient genome editing at the target site.The editing efficiency resulted in a 52.73%nymph hatching rate and 66.67%of the G0 adults were mosaic mutants. The passaging strategy of the mutant lines is shown here. The excess homozygous eggs were cryopreserved to improve the utilization rate of the homozygotes.Although the hatching rate of the cryopreserved eggs was reduced with the extension of cryopreservation time, the remedial actions such as applying filter paper fragments and recovering these preserved eggs with a gradient of rising temperature were helpful for the resuscitation. The most important thing is to try to limit the mechanical damage caused by microinjection. This procedure can be used as a reference for the attaining other insects.Part of some details such as methods of egg collection, injection and the crop should be modified. This technique can also be helpful to study metamorphosis and the environmental adaptation of the insects and the construction of homozygous locust mutants accelerated the research in this field.

Research

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