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 HÃƒÂ¼bner 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...

Watch this Scientific Journal Video about Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera HÃƒÂ¼bner at JoVE.com

Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera HÃƒÆ’Ã‚Â¼bner

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 ...

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