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

Qiang Yan; Yingying He; Yang Yue; Luyao Jie; Tingmei Wen; Yumiao Zhao; Min Zhang; Tingting Zhang

doi:10.3791/63629

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

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

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10:07 min

March 16, 2022

DOI: 10.3791/63629-v

Qiang Yan*¹, Yingying He*^2,3, Yang Yue^2,3, Luyao Jie¹, Tingmei Wen^2,3, Yumiao Zhao^2,3, Min Zhang², Tingting Zhang²

¹State Key Laboratory of Cotton Biology, Key Laboratory of Plant Stress Biology, School of Life Sciences,Henan University, ²Research Institute of Applied Biology,Shanxi University, ³School of Life Science,Shanxi University

Overview

This study demonstrates a systematic optimization of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants using the migratory locust as a model system. Additionally, it details a method for embryo cryopreservation and resuscitation, highlighting important protocols for genetic manipulation and preservation of locust eggs.

Key Study Components

Research Area

Genetic engineering of locusts
CRISPR/Cas9 technology
Cryopreservation methods

Background

The migratory locust is a significant agricultural pest.
Efficient protocols are required for genetic studies and pest management.
CRISPR/Cas9 is a powerful tool for genome editing in various organisms.

Methods Used

CRISPR/Cas9 protocol for constructing homozygous mutants
Collection and handling of locust eggs
Microinjection techniques and cryopreservation

Main Results

Achieved a gene editing efficiency leading to a significant nymph hatching rate.
Documented successful cryopreservation and resuscitation processes.
Establish a viable method for maintaining homozygous mutant lines.

Conclusions

This study provides valuable protocols for further research in insect genetics.
Impacts agricultural science and pest management strategies.

Frequently Asked Questions

What is the main focus of the study?

The study focuses on optimizing CRISPR/Cas9 methods for creating homozygous locust mutants and methods for cryopreservation of locust eggs.

How does CRISPR/Cas9 contribute to this research?

CRISPR/Cas9 allows for precise genetic modifications in the locusts, which can help understand genetic functions and traits.

What are the benefits of cryopreservation in this context?

Cryopreservation enables long-term storage of genetic materials, facilitating studies in genetics and breeding.

What model organism is used in this research?

The migratory locust is the primary model organism used to study genetic modifications and preservation techniques.

What are the potential applications of this research?

This research has implications for pest management and the study of insect biology.

What was the outcome concerning mutation rates?

The study found a significant hatching rate from modified embryos, indicating effective gene editing.

How does this research impact agricultural science?

By improving understanding of locust genetics, this research could lead to better management strategies for locust populations, which are significant agricultural pests.

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 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.

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Homozygous Mutants Migratory Locust CRISPR/Cas9 Technology Gene Editing Injection Needles Ribonucleoprotein Egg Collection Mutant Screening Microinjection Cas9 Protein Single Guide RNA Injection Parameters Incubation Agricultural Testing