Name: embryo microinjection and knockout mutant identification of crispr/cas9 genome-edited helicoverpa armigera (h&#252;bner)
Uploaded: 2021-07-01T13:00:00
Description: Watch this Scientific Journal Video about Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera HÃ¼bner at JoVE.com

This work was supported by National Natural Science Foundation of China (31725023, 31861133019 to GW, and 31171912 to CY).

1. Design of gene-specific primers and preparation of sgRNA
<ol>
	<li>Verify a conserved genomic region in the gene of interest through PCR amplification and sequencing analyses. Amplify the target gene from the genome DNA of H. armigera and distinguish the exons and introns. 
	NOTE: The sequence specificity of the guide site is necessary to avoid off-target gene editing. Search possible guide sites in the exons are close to the 5&#39; UTR of the gene. Then, it is important to make sure that the gene is c...

<ol>
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The application of the CRISPR/Cas9 system has provided powerful technical support for the analysis of gene function and interaction among various genes. The detailed protocol we present here demonstrates the generation of a homozygote mutant in H. armigera via CRISPR/Cas9 genome editing. This reliable procedure provides a straightforward way for directed gene mutagenesis in H. armigera.
The choice of CRISPR target sites could affect the mutagenesis efficiency<sup class="xref"...

The application of genome editing technology provides an efficient tool to achieve target-gene mutants in diverse species. The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) system provides a novel method to manipulate genomes1. The CRISPR/Cas9 system consists of a guide RNA (gRNA) and the Cas9 endonuclease2,3, while the gRNA can be further divided into two parts, a target complementary CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). The gRNA integrates with Cas9 endonuclease and forms a ribonucleoprotein (RNP). With the gRNA, Cas9 endonuclease can be directed to a specific site of the genome via base complementation. The RuvC and HNH domains of the Cas9 cleave the target site of the genome three bases before the protospacer-adjacent motif (PAM) sequence and create a double-strand break (DSB). The DNA cleavage can then be repaired through two mechanisms, non-homologous end joining (NHEJ) or homology-directed repair (HDR)4. Repair of the DSB introduces insertions or deletions as a way to inactivate the targeted gene, potentially causing a complete loss of gene function. Hence, the hereditable and specificity of the CRISPR/Cas9 system make it a robust method to characterize gene functions in vivo and analyze gene interactions5.
With numerous merits, the CRISPR/Cas9 system has been applied to various fields, including biomedicine6,7, gene therapy8,9, and agriculture10,11,12, and has been used for various biological systems including microorganisms13, plants14,15, nematodes16 , and mammals17. In invertebrates, many insect species have been subjected to CRISPR/Cas9 genome editing, such as the fruit fly Drosophila melanogaster and beyond18,19,20,21,22.
Helicoverpa armigera is one of the most destructive pests worldwide23, and damages numerous crops, including cotton, soybean, and sorghum24,25. With the development of sequencing technology, the genome of H. armigera, as well as that of a range of Lepidoptera insect species, have been sequenced completely26,27,28,29. A large number of resistance and olfactory receptor genes have been identified and characterized from these insects in recent years19,27,28,29. Some resistance-related genes have been identified in H. armigera, such as the genes encoding for cadherin30, an ATP-binding cassette transporter31,32, as well as HaTSPAN133. Knockout of these genes using CRISPR/Cas9 technology results in a high level of resistance to Bacillus thuringiensis (BT) toxin in susceptible strains. Also, Chang et al. (2017) knocked out a pheromone receptor, which validated its significant function in mating time regulation19. These reports suggest that CRISPR/Cas9 can act as an effective tool to study gene function in vivo in insect systems. However, a detailed procedure for CRISPR/Cas9 modification in insect systems remains incomplete, which limits its application range in insect functional genomics.
Here, we present a protocol for knocking out a functional gene in H. armigera using the CRISPR/Cas9 system. A detailed step-by-step protocol is provided, including the design and preparation of gene-specific primers for gRNA production, embryo collection, microinjection, insect rearing, and mutant identification. This protocol serves as a valuable reference to manipulate any functional genes in H. armigera and can be extended to other Lepidoptera species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2kb DNA ladder</td>
			<td>TransGen Biotech</td>
			<td>BM101</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Glass</td>
			<td>World Precision Instrucments</td>
			<td>504949</td>
			<td>referred to as &#34;capillary glass&#34; in the protocol</td>
		</tr>
		<tr>
			<td>Double Sided Tape</td>
			<td>Minnesota Mining and Manufacturing Corporation</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf FemtoJet 4i Microinjector</td>
			<td>Eppendorf Corporate</td>
			<td>E5252000021</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf InjectMan 4 micromanipulator</td>
			<td>Eppendorf Corporate</td>
			<td>5192000051</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microloader Pipette Tips</td>
			<td>Eppendorf Corporate</td>
			<td>G2835241</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneArt Precision gRNA Synthesis Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A29377</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slide</td>
			<td>Sail Brand</td>
			<td>7105</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Microscope</td>
			<td>Olympus Corporation</td>
			<td>SZX16</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeSTAR HS (Premix)</td>
			<td>Takara Biomedical Technology</td>
			<td>R040</td>
			<td>used for mutant detection</td>
		</tr>
		<tr>
			<td>Sutter Micropipette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>TIANamp Genomic DNA Kit</td>
			<td>TIANGEN Corporate</td>
			<td>DP304-03</td>
			<td></td>
		</tr>
		<tr>
			<td>TrueCut Cas9 Protein v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>A36499</td>
			<td></td>
		</tr>
	</tbody>
</table>

embryo microinjection and knockout mutant identification of crispr/cas9 genome-edited helicoverpa armigera (h&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.

This protocol provides detailed steps for obtaining gene knock-out lines of H. armigera using CRISPR/Cas9 technology. The representative results obtained by this protocol are summarized for gDNA selection, embryo collection and injection, insect rearing, and mutant detection.
In this study, the target site of our gene of interest was located in its second exon (Figure 2A). This site was hig...

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Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera H&#252;bner

Presented here is a protocol of Helicoverpa armigera (H&#252;bner) embryo microinjection and knockout mutant identification created by CRISPR/Cas9 genome editing. Mutant insects enable further research of gene function and interaction among different genes in vivo.

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (H&#252;bner)

The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional ...

Cotton bollworm is one of the most destructive pests worldwide. Genome editing with the CRISPR/Cas9 technology enables further research of gene function and interaction among different genes in this organism. The two main advantages of this technique are that it has high specificity and is hereditable.In order to choose the sgRNA targets, go to the CRISPR online website to search for possible guide sites and the exons close to the five prime untranslated region of the gene. Input the exon sequence into the text box and select the target genome to Helicoverpa armigera. Choose the PAM option for 20 BPNGG and leave the other settings on the default parameters, according to the user manuals of the website.Compare the predicted guide sequences from the software and choose a guide sequence of 20 base pairs containing one or two guanines near the five prime untranslated region with the highest predicted efficiency and fewest mismatches to improve the editing efficiency and reduce off-target editing. For embryo preparation, select 50 healthy individuals from three-day-old males and two-day-old females, and mix them in a clean net cage. Place a piece of moist cotton containing 10%sugar solution in the cage.Cover the net cages with gauze and fix the gauze with a rubber band. Spray water onto the gauze to keep it moist. Cut a black cloth into an appropriate size and replace the moist gauze with this black cloth to enable free ova-position for 30 minutes.Paste double-sided tape onto a microscope slide. Using forceps, paste the patches with eggs in a row on the surface of the double-sided tape. Press the margin of each patch to make sure they stick firmly to the tape.Collect 50 to 100 eggs per microscope slide. Before microinjection, keep the microscope slide on ice to delay the development of embryos. Prepare the needle by pulling a capillary glass using a micro pipette puller as described in the text manuscript.Grind the needle tip using a micro grinder to a sharp-edged tip. Prepare injection solutions by adding two microliters of commercialized Cas9 protein and sgRNA to RNace-free water in a PCR tube to obtain 10 microliter volume mixture. Mix well by pipetting and put it on ice.Set the parameters of the electronic microinjector. Load two microliters of the mixture into a needle using a microloader pipette tip, exhausting as much residual air in the tip of the needle as possible. Connect the injection needle to a micromanipulator and ensure a tight connection between the two parts.Place a slide in a Petri dish and put it on the stage of the microscope. Carefully insert the needle tip into the top hemisphere of an embryo at a 45 degree angle. Press the pedal to deliver the mixture into the embryo, leading to a slight expansion of the embryo.Retract the needle immediately from the embryo and move the Petri dish with one hand until the next embryo is in proximity to the needle. Inject at least 300 embryos using the same procedure to ensure a sufficient hatching amount. Cover the lid of the Petri dishes after injection.When the surface color of embryos has darkened, put artificial diet in the Petri dish around the microscope slide. Prepare 24-well culture plates and fill each well to 1/3 of its volumetric capacity with artificial diet. Pick out hatching larva using a small paint brush and transfer them to the 24-well culture plate, such that one well has one larva.When larva grow to the third instar stage, transfer them to a new glass ductile ethrae. Transfer the newly enclosed G-zero male adults and wild-type female adults into a fresh net cage at an equal ratio. Supply them with 10%sugar solution dropped in cotton balls.Rear insects using routine methods until the pupation of G-one. Use forceps to carefully remove a hind leg and put each leg in a Lysing Matrix tube. Homogenize the hind leg using a tissue homogenizer.Extract the genomic DNA of the homogenized sample using a commercial gDNA extraction kit according to the manufacturer's instructions. Amplify the gene segment using genotyping primers and the PCR reaction conditions from the text manuscript. Confirm the genotype with a gene-sequencing service.Put G-one individuals of the same genotype in one net cage, self cross the G-one progenys and continue to screen using the same methods. The target site of the gene of interest was located in its second exon. This site was highly conserved and the target band fragment of synthesized sgRNA was confirmed using agarose gel electrophoresis.The mutant detection of a single sgRNA target was performed by sequencing the PCR products from G-one parental units. The effectiveness of using non-overlapping sgRNA pairs across different exons was demonstrated as the large deletion of the mutants was easily distinguished from the wild-type pans. After obtaining a certain number of mutant individuals, electrophysiological or behavioral experiments can be performed to clarify gene function and interaction among different genes.

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