Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner)

Summary

Presented here is a protocol of Helicoverpa armigera (Hübner) 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.

Abstract

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.

Introduction

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 endonuclease^2,3, 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)....

Protocol

1. Design of gene-specific primers and preparation of sgRNA

1. 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' UTR of the gene. Then, it is important to make sure that the gene is completely nonfunctional. A summary of the flow path for the preparation of sgRNA is illustrated in Figure 1.
2. C....

Results

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.......

Discussion

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

Materials

Name	Company	Catalog Number	Comments
2kb DNA ladder	TransGen Biotech	BM101
Capillary Glass	World Precision Instrucments	504949	referred to as "capillary glass" in the protocol
Double Sided Tape	Minnesota Mining and Manufacturing Corporation	665
Eppendorf FemtoJet 4i Microinjector	Eppendorf Corporate	E5252000021
Eppendorf InjectMan 4 micromanipulator	Eppendorf Corporate	5192000051
Eppendorf Microloader Pipette Tips	Eppendorf Corporate	G2835241
GeneArt Precision gRNA Synthesis Kit	Thermo Fisher Scientific	A29377
Microscope Slide	Sail Brand	7105
Olympus Microscope	Olympus Corporation	SZX16
PrimeSTAR HS (Premix)	Takara Biomedical Technology	R040	used for mutant detection
Sutter Micropipette Puller	Sutter Instrument Company	P-1000
TIANamp Genomic DNA Kit	TIANGEN Corporate	DP304-03
TrueCut Cas9 Protein v2	Thermo Fisher Scientific	A36499