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

Summary

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.

Abstract

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, 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.

Introduction

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 sciences^2,3,4,5,6. 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 ....

Protocol

1. Target site selection and sgRNA design

1. 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.
2. Compare the sequence of the interested gene to its genomic DNA sequence to distinguish the exon and intron regions.
3. Select a candidate region for target site design based on the research purpose. Design primer pairs to amplify the candidate region fragment and verify its wild-type sequence by sequencing analysis of the PCR product.
   NOTE: There are ....

Results

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 synthesis and verification (Figure 1A), egg collection and injection, mutant screening and passaging, cryopreservation, and resuscitation of the homozygous eggs.

Discussion

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.......

Materials

List of materials used in this article

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